Structured Review

Beckman Coulter ovcar5
DNA damage response and cell cycle analysis after CHEK1 and IKKε inhibition (A) Western blotting for DNA damage response. Phosphorylated CHEK1, total CHEK1, and γ-H2A.X were measured by Western blotting after 24 hour treatment at final concentrations of 2 μM (BX795) and/or 0.5 μM (PF477736) prepared in fresh medium. GAPDH and H2A.X were used as loading controls. (B) Cell cycle was analyzed after pharmacological intervention in <t>Ovcar5.</t> Cells were treated for 16 hours at final concentrations of 2 μM (BX795) and/or 0.5 μM (PF477736) prepared in fresh medium. G 0 /G 1 , S, and G 2 /M phases were measured based on staining of APC-BrdU and 7-AAD by flow cytometry. (C) Western blotting for cell cycle regulators. The treatment conditions and total cellular lysates preparations are same as in Figure 4E–F .
Ovcar5, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 89/100, based on 1232 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
https://www.bioz.com/result/ovcar5/product/Beckman Coulter
Average 89 stars, based on 1232 article reviews
Price from $9.99 to $1999.99
ovcar5 - by Bioz Stars, 2020-09
89/100 stars

Images

1) Product Images from "Loss of compensatory pro-survival and anti-apoptotic modulator, IKKε, sensitizes ovarian cancer cells to CHEK1 loss through an increased level of p21"

Article Title: Loss of compensatory pro-survival and anti-apoptotic modulator, IKKε, sensitizes ovarian cancer cells to CHEK1 loss through an increased level of p21

Journal: Oncotarget

doi:

DNA damage response and cell cycle analysis after CHEK1 and IKKε inhibition (A) Western blotting for DNA damage response. Phosphorylated CHEK1, total CHEK1, and γ-H2A.X were measured by Western blotting after 24 hour treatment at final concentrations of 2 μM (BX795) and/or 0.5 μM (PF477736) prepared in fresh medium. GAPDH and H2A.X were used as loading controls. (B) Cell cycle was analyzed after pharmacological intervention in Ovcar5. Cells were treated for 16 hours at final concentrations of 2 μM (BX795) and/or 0.5 μM (PF477736) prepared in fresh medium. G 0 /G 1 , S, and G 2 /M phases were measured based on staining of APC-BrdU and 7-AAD by flow cytometry. (C) Western blotting for cell cycle regulators. The treatment conditions and total cellular lysates preparations are same as in Figure 4E–F .
Figure Legend Snippet: DNA damage response and cell cycle analysis after CHEK1 and IKKε inhibition (A) Western blotting for DNA damage response. Phosphorylated CHEK1, total CHEK1, and γ-H2A.X were measured by Western blotting after 24 hour treatment at final concentrations of 2 μM (BX795) and/or 0.5 μM (PF477736) prepared in fresh medium. GAPDH and H2A.X were used as loading controls. (B) Cell cycle was analyzed after pharmacological intervention in Ovcar5. Cells were treated for 16 hours at final concentrations of 2 μM (BX795) and/or 0.5 μM (PF477736) prepared in fresh medium. G 0 /G 1 , S, and G 2 /M phases were measured based on staining of APC-BrdU and 7-AAD by flow cytometry. (C) Western blotting for cell cycle regulators. The treatment conditions and total cellular lysates preparations are same as in Figure 4E–F .

Techniques Used: Cell Cycle Assay, Inhibition, Western Blot, Staining, Flow Cytometry, Cytometry

Validation of CHEK1 lethal effects in IKKε-mtached Ovcar5 cells (A) The knockdown levels of CHEK1 by 3 different shRNAs were examined by Western blot analysis. GAPDH was used as a loading control. (B) Double-positive cells after IKKε and CHEK1 shRNA knockdown were monitored by flow cytometry after two rounds of infection. Dot plots at 7 doubling times were shown. IKKε shRNA constructs co-express LYT2 (mouse CD8α) which was detected by an antibody conjugated to APC; CHEK1 shRNA constructs co-expressed GFP. (C) Two previously validated IKKε shRNAs were used. Either IKKε #1 or IKKε #2 shRNA (in pRSM-LYT2 vector) was co-infected with one of 3 different CHEK1 shRNAs (in pRSMX-PG vector) into Ovcar5 cells. The changes in double-positive cells were measured and calculated relative to 4 doublings after infection, at which GFP signal is maximal. The average number from 3 different CHEK1 shRNA in either negative shRNA or IKKε shRNA were calculated during indicated time courses. The significant p value was calculated by a 2-sided student's t-test. (D) A2780 IKKε-matched cell lines were established and shRNA library screening performed in the same manner as in Ovcar5. Using a cut-off with a fold change of ≤ 0.7 with a p -value of ≤ 0.05 shown in bold, 3 different CHEK1 shRNAs were identified at the different time points. (E) The expression levels of IKKε and CHEK1 proteins (left) and mRNAs (right) were examined upon IKKε knockdown in two cell lines. GAPDH was used as a loading control for immune blotting. The area of each band was quantified by ImageJ and normalized by GAPDH, and expressed as the level relative to its negative control. The mRNA levels were quantified by real-time PCR, normalized by that of GAPDH, and the expression was calculated relative to that in negative shRNA infected cells.
Figure Legend Snippet: Validation of CHEK1 lethal effects in IKKε-mtached Ovcar5 cells (A) The knockdown levels of CHEK1 by 3 different shRNAs were examined by Western blot analysis. GAPDH was used as a loading control. (B) Double-positive cells after IKKε and CHEK1 shRNA knockdown were monitored by flow cytometry after two rounds of infection. Dot plots at 7 doubling times were shown. IKKε shRNA constructs co-express LYT2 (mouse CD8α) which was detected by an antibody conjugated to APC; CHEK1 shRNA constructs co-expressed GFP. (C) Two previously validated IKKε shRNAs were used. Either IKKε #1 or IKKε #2 shRNA (in pRSM-LYT2 vector) was co-infected with one of 3 different CHEK1 shRNAs (in pRSMX-PG vector) into Ovcar5 cells. The changes in double-positive cells were measured and calculated relative to 4 doublings after infection, at which GFP signal is maximal. The average number from 3 different CHEK1 shRNA in either negative shRNA or IKKε shRNA were calculated during indicated time courses. The significant p value was calculated by a 2-sided student's t-test. (D) A2780 IKKε-matched cell lines were established and shRNA library screening performed in the same manner as in Ovcar5. Using a cut-off with a fold change of ≤ 0.7 with a p -value of ≤ 0.05 shown in bold, 3 different CHEK1 shRNAs were identified at the different time points. (E) The expression levels of IKKε and CHEK1 proteins (left) and mRNAs (right) were examined upon IKKε knockdown in two cell lines. GAPDH was used as a loading control for immune blotting. The area of each band was quantified by ImageJ and normalized by GAPDH, and expressed as the level relative to its negative control. The mRNA levels were quantified by real-time PCR, normalized by that of GAPDH, and the expression was calculated relative to that in negative shRNA infected cells.

Techniques Used: Western Blot, shRNA, Flow Cytometry, Cytometry, Infection, Construct, Plasmid Preparation, Library Screening, Expressing, Negative Control, Real-time Polymerase Chain Reaction

Pharmacological intervention of IKKε and CHEK1 in ovarian cancer cell lines (A) IKKε inhibitor (BX795) and CHEK1 inhibitor (PF477736) sensitivities were measured by XTT assay. Cells were seeded at 1000 cells/well in 50 μl and 4 replicates 16-20 hours prior to the addition of the drug. XTT assay was performed 3 days later upon drug treatment. The viability was calculated relative to no drug treated samples. (B) Cellular viability assay was performed in 2-fold serial dilutions of PF477736 (range 0 – 0.4 μM) in triplicate in a 96 well matrix format, and then cellular death (Fa; fraction affected) was calculated from XTT based viability fractions. IC50 was calculated using CompuSyn software. Shown are the results in the presence of BX795 (circle; 1.25 μM, except 0.625 μM for A2780) and the absence of the IKKε inhibitor (square). (C) IKKε and CHEK1 doubly depleted Ovcar5 and A2780 cells maintained in selective media (25 μg/ml of mycophenolic acid and 2 μg/ml of puromycin) were seeded in 96-well plates 24 hours prior to the addition of CHEK1 inhibitor PF477736. XTT assay was performed 3 days later upon drug treatment. Statistical tests were 2-sided between shNeg and shIKKε in the presence of CHEK1 inhibitor. (D) The relative cell viability was calculated based on no drug treatment, and is shown at the following suboptimal concentrations of BX795/PF477736: 1.25 μM/0.2 μM (Ovcar5, HeyA8), 1.25 μM/0.1 μM (Ovcar8, Skov3), 0.625 μM/0.1 μM (A2780), and 0.625 μM/0.05 μM (Igrov1). SD was calculated from 3 replicates. XTT assay was performed 3 days after treatment. (E) Caspase-3/7 activity was measured by Caspase-Glo assay after exposure to BX795 (2 μM) and/or PF477736 (0.5 μM) for 24 hours. Mean values ± SD of 3 replicates are shown and the data represent 4 independent experiments. (F) The cleavage of apoptotic indicator PARP-1 was examined by Western blot analysis. Cells were treated for 24 hours as the same conditions as in Figure 4E and harvested for total cell lysate preparation. Total PARP1 is shown as a loading control.
Figure Legend Snippet: Pharmacological intervention of IKKε and CHEK1 in ovarian cancer cell lines (A) IKKε inhibitor (BX795) and CHEK1 inhibitor (PF477736) sensitivities were measured by XTT assay. Cells were seeded at 1000 cells/well in 50 μl and 4 replicates 16-20 hours prior to the addition of the drug. XTT assay was performed 3 days later upon drug treatment. The viability was calculated relative to no drug treated samples. (B) Cellular viability assay was performed in 2-fold serial dilutions of PF477736 (range 0 – 0.4 μM) in triplicate in a 96 well matrix format, and then cellular death (Fa; fraction affected) was calculated from XTT based viability fractions. IC50 was calculated using CompuSyn software. Shown are the results in the presence of BX795 (circle; 1.25 μM, except 0.625 μM for A2780) and the absence of the IKKε inhibitor (square). (C) IKKε and CHEK1 doubly depleted Ovcar5 and A2780 cells maintained in selective media (25 μg/ml of mycophenolic acid and 2 μg/ml of puromycin) were seeded in 96-well plates 24 hours prior to the addition of CHEK1 inhibitor PF477736. XTT assay was performed 3 days later upon drug treatment. Statistical tests were 2-sided between shNeg and shIKKε in the presence of CHEK1 inhibitor. (D) The relative cell viability was calculated based on no drug treatment, and is shown at the following suboptimal concentrations of BX795/PF477736: 1.25 μM/0.2 μM (Ovcar5, HeyA8), 1.25 μM/0.1 μM (Ovcar8, Skov3), 0.625 μM/0.1 μM (A2780), and 0.625 μM/0.05 μM (Igrov1). SD was calculated from 3 replicates. XTT assay was performed 3 days after treatment. (E) Caspase-3/7 activity was measured by Caspase-Glo assay after exposure to BX795 (2 μM) and/or PF477736 (0.5 μM) for 24 hours. Mean values ± SD of 3 replicates are shown and the data represent 4 independent experiments. (F) The cleavage of apoptotic indicator PARP-1 was examined by Western blot analysis. Cells were treated for 24 hours as the same conditions as in Figure 4E and harvested for total cell lysate preparation. Total PARP1 is shown as a loading control.

Techniques Used: XTT Assay, Cell Viability Assay, Software, Activity Assay, Caspase-Glo Assay, Western Blot

The effect of IKKε manipulation on p21 level and its effect on survival of ovarian cancer cells (A) Ovcar5 and A2780 were depleted of IKKε or negative control and maintained under selection (25 μg/ml of mycophenolic acid). Total protein lysates were prepared to examine p21 level and the knockdown of IKKε. GAPDH was used as a loading control. (B) Caov3-wtIKKε and Caov3-IKKε K38A were maintained in selective media (100 μg/ml of G418) and total protein lysates were prepared for the protein analysis. (C) Cytoplasimc and nuclear protein fractionation was prepared using Ovcar5 and A2780 depleted for IKKε (shRNA#1) or control and maintained in culture after magnetic beads purification for LYT2 surface selection marker. (D) Ovcar5 cells were treated for 4 hours with IKKε inhibitor (BX795, 2 μM) and/or CHEK1 inhibitor (PF477736, 0.5 μM) prepared in fresh medium prior to cellular fractionation. GAPDH and Topoisomerase II were used as cytosolic and nuclear markers, respectively. (E) Cells transfected with either siNeg or si p21 were seeded at 2000 cells/well in 50 μl in 3 replicates and 8 hours later PF477736 was added in the absence or presence of BX795 (0.3 μM). XTT assay was performed 2 and 3 days later upon drug treatment. The viability was calculated relative to no drug and siNeg treated samples. The significant p values between siNeg and si p21 samples were calculated by a 2-sided student's t-test. (F) After 16 hours siRNA transfection, cells were seeded and treated for 16 hours with IKKε inhibitor (BX795, 0.3 μM) and/or CHEK1 inhibitor (PF477736, 0.4 μM) in 4 replicates. The caspase3/7 activities were shown relative to untreated siNeg samples from 4 replicates. The significant p values between siNeg and si p21 samples were calculated by a 2-sided student's t-test.
Figure Legend Snippet: The effect of IKKε manipulation on p21 level and its effect on survival of ovarian cancer cells (A) Ovcar5 and A2780 were depleted of IKKε or negative control and maintained under selection (25 μg/ml of mycophenolic acid). Total protein lysates were prepared to examine p21 level and the knockdown of IKKε. GAPDH was used as a loading control. (B) Caov3-wtIKKε and Caov3-IKKε K38A were maintained in selective media (100 μg/ml of G418) and total protein lysates were prepared for the protein analysis. (C) Cytoplasimc and nuclear protein fractionation was prepared using Ovcar5 and A2780 depleted for IKKε (shRNA#1) or control and maintained in culture after magnetic beads purification for LYT2 surface selection marker. (D) Ovcar5 cells were treated for 4 hours with IKKε inhibitor (BX795, 2 μM) and/or CHEK1 inhibitor (PF477736, 0.5 μM) prepared in fresh medium prior to cellular fractionation. GAPDH and Topoisomerase II were used as cytosolic and nuclear markers, respectively. (E) Cells transfected with either siNeg or si p21 were seeded at 2000 cells/well in 50 μl in 3 replicates and 8 hours later PF477736 was added in the absence or presence of BX795 (0.3 μM). XTT assay was performed 2 and 3 days later upon drug treatment. The viability was calculated relative to no drug and siNeg treated samples. The significant p values between siNeg and si p21 samples were calculated by a 2-sided student's t-test. (F) After 16 hours siRNA transfection, cells were seeded and treated for 16 hours with IKKε inhibitor (BX795, 0.3 μM) and/or CHEK1 inhibitor (PF477736, 0.4 μM) in 4 replicates. The caspase3/7 activities were shown relative to untreated siNeg samples from 4 replicates. The significant p values between siNeg and si p21 samples were calculated by a 2-sided student's t-test.

Techniques Used: Negative Control, Selection, Fractionation, shRNA, Magnetic Beads, Purification, Marker, Cell Fractionation, Transfection, XTT Assay

Human kinome shRNA screening (A) Dual shRNA screening procedure (B) The magnetic beads-based purified cells followed by shRNA library infection were maintained for indicated times upon completion of puromycin selection for 4 days, and then harvested for Western blotting in four biological replicates. For quantification, the signals were quantified by ImageJ software. IKKε expression was normalized by GAPDH level. The statistical significance was determined by 2-sided t-test. (C) The purity of Ovcar5 CD8α-positive cells (IKKε-matched cell line pairs) was measured by FACS at 13 days after beads purification. (D) The shRNA library vectors co-expressing GFP were introduced into the pseudo-isogenic IKKε-control and -depleted cell line pair. The purity of shRNA library in Ovcar5 cells was measured by GFP expression 2 days after completion of puromycin selection.
Figure Legend Snippet: Human kinome shRNA screening (A) Dual shRNA screening procedure (B) The magnetic beads-based purified cells followed by shRNA library infection were maintained for indicated times upon completion of puromycin selection for 4 days, and then harvested for Western blotting in four biological replicates. For quantification, the signals were quantified by ImageJ software. IKKε expression was normalized by GAPDH level. The statistical significance was determined by 2-sided t-test. (C) The purity of Ovcar5 CD8α-positive cells (IKKε-matched cell line pairs) was measured by FACS at 13 days after beads purification. (D) The shRNA library vectors co-expressing GFP were introduced into the pseudo-isogenic IKKε-control and -depleted cell line pair. The purity of shRNA library in Ovcar5 cells was measured by GFP expression 2 days after completion of puromycin selection.

Techniques Used: shRNA, Magnetic Beads, Purification, Infection, Selection, Western Blot, Software, Expressing, FACS

2) Product Images from "Whole genome sequencing of Plasmodium falciparum from dried blood spots using selective whole genome amplification"

Article Title: Whole genome sequencing of Plasmodium falciparum from dried blood spots using selective whole genome amplification

Journal: Malaria Journal

doi: 10.1186/s12936-016-1641-7

Selective whole genome amplification (sWGA) enrichment. Simulated clinical samples comprising 96% human DNA and 4% P. falciparum DNA (3D7) were amplified using either WGA or sWGA. Amplified samples were sequenced to determine the proportion of reads mapping to human or P. falciparum reference genomes
Figure Legend Snippet: Selective whole genome amplification (sWGA) enrichment. Simulated clinical samples comprising 96% human DNA and 4% P. falciparum DNA (3D7) were amplified using either WGA or sWGA. Amplified samples were sequenced to determine the proportion of reads mapping to human or P. falciparum reference genomes

Techniques Used: Whole Genome Amplification, Amplification

Core genome coverage profile. Coverage depth of chromosome 1 by leucodepleted and unamplified (VB), whole genome amplified (WGA) and selective whole genome amplified (sWGA) DNA of P. falciparum strain 3D7. Black horizontal line shows positions corresponding to the core genome and red vertical line shows the centromere
Figure Legend Snippet: Core genome coverage profile. Coverage depth of chromosome 1 by leucodepleted and unamplified (VB), whole genome amplified (WGA) and selective whole genome amplified (sWGA) DNA of P. falciparum strain 3D7. Black horizontal line shows positions corresponding to the core genome and red vertical line shows the centromere

Techniques Used: Amplification, Whole Genome Amplification

3) Product Images from "Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer"

Article Title: Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer

Journal: GigaScience

doi: 10.1093/gigascience/gix043

Microbial structure of the mock communities. ( A and B ) Microbial species and respective relative proportions determined to be present in the HM782D and D6305 mock communities, respectively, following the analysis of raw data obtained from rrn amplicon sequencing in MinION™ and chemistry R9. ( C and D ) Comparative analysis of the expected microbial species and proportions against the data obtained after mapping reads generated by a 4.5-kbp amplicon PCR and sequenced in the MinION™ device with R9 and R9.4 chemistries, for HM782D and D6305, respectively. ( E ) Linear correlation analysis of relative read proportions obtained for all bacterial species present in HM872D and D6305 mock communities with R9 and R9.4 chemistries.
Figure Legend Snippet: Microbial structure of the mock communities. ( A and B ) Microbial species and respective relative proportions determined to be present in the HM782D and D6305 mock communities, respectively, following the analysis of raw data obtained from rrn amplicon sequencing in MinION™ and chemistry R9. ( C and D ) Comparative analysis of the expected microbial species and proportions against the data obtained after mapping reads generated by a 4.5-kbp amplicon PCR and sequenced in the MinION™ device with R9 and R9.4 chemistries, for HM782D and D6305, respectively. ( E ) Linear correlation analysis of relative read proportions obtained for all bacterial species present in HM872D and D6305 mock communities with R9 and R9.4 chemistries.

Techniques Used: Amplification, Sequencing, Generated, Polymerase Chain Reaction

4) Product Images from "Driving the scalability of DNA-based information storage systems"

Article Title: Driving the scalability of DNA-based information storage systems

Journal: bioRxiv

doi: 10.1101/591594

Physical file separations in DENSE storage rescue the decreased sequencing efficiency experienced by high-capacity databases. a , A library of five files was ordered and analyzed using NGS to confirm an even file distribution. b , File 3 strands were enriched over increasingly higher capacity backgrounds of non-specific DNA strands using 30 cycles of random-access PCR. Random access failed to enrich File 3 to above 50% of the total sample once the background capacity reached 31.1 GB, as measured by quantitative PCR. c , DENSE physically extracts a file (orange) from the database so only its strands are sequenced. A primer functionalized with a chemical handle (yellow diamond) is used to execute one emulsion PCR cycle to create chemically labeled copies of the desired file’s strands. Functionalized magnetic beads (brown) that bind to the chemical handle are added to the sample. The desired file is bound to the bead, and the unbound solution containing the original database is removed and saved for future reuse. The bound file is then eluted from the bead. d , After biotin-streptavidin file extractions, the remaining solution still contained all files while the target files were enriched and physically separated, as measured by next generation sequencing. By mapping sequencing reads to the original file sequences, all targeted data were confirmed recovered. The target file was retained in the supernatant containing the database and was able to be copied and extracted again. File 1 was extracted three sequential times, and File 2 was extracted from the solution remaining after an initial extraction of File 1. e , File extractions using fluorescein, digoxigenin, and polyA(25) as chemical handles also successfully separated target files from the database. f , A large-scale background mimicking diverse data was created using error prone PCR 13 to mutagenize and amplify File 1. g , Random access was compared directly to chemical handle extractions. File 3 strands, with a starting fraction of 0.03% of the total number of strands, were enriched over a high-capacity background equivalent to 5.53 TB of undesired, non-specific strands using either random access (black) or PCR followed by chemical handle primer extractions (blue, green, purple or pink). After 5, 15, and 30 cycles of PCR (random access), enrichment of File 3 was 0.0%, 0.0%, and 1.69% of the total sample, respectively. After biotin-modified PCR followed by extraction, the enrichment of File 3 was 0.2%, 87.5%, and 100% of the total sample, respectively. After fluorescein-modified PCR followed by extraction, the enrichment of File 3 was 0.1%, 49.6%, and 100% of the total sample, respectively. After digoxigenin-modified PCR followed by extraction, the enrichment of File 3 was 0.2%, 14.2%, and 100% of the total sample, respectively. After poly(A)-25-modified PCR followed by extraction, the enrichment of File 3 was 0.09%, 0.47%, and 100% of the total sample, respectively.
Figure Legend Snippet: Physical file separations in DENSE storage rescue the decreased sequencing efficiency experienced by high-capacity databases. a , A library of five files was ordered and analyzed using NGS to confirm an even file distribution. b , File 3 strands were enriched over increasingly higher capacity backgrounds of non-specific DNA strands using 30 cycles of random-access PCR. Random access failed to enrich File 3 to above 50% of the total sample once the background capacity reached 31.1 GB, as measured by quantitative PCR. c , DENSE physically extracts a file (orange) from the database so only its strands are sequenced. A primer functionalized with a chemical handle (yellow diamond) is used to execute one emulsion PCR cycle to create chemically labeled copies of the desired file’s strands. Functionalized magnetic beads (brown) that bind to the chemical handle are added to the sample. The desired file is bound to the bead, and the unbound solution containing the original database is removed and saved for future reuse. The bound file is then eluted from the bead. d , After biotin-streptavidin file extractions, the remaining solution still contained all files while the target files were enriched and physically separated, as measured by next generation sequencing. By mapping sequencing reads to the original file sequences, all targeted data were confirmed recovered. The target file was retained in the supernatant containing the database and was able to be copied and extracted again. File 1 was extracted three sequential times, and File 2 was extracted from the solution remaining after an initial extraction of File 1. e , File extractions using fluorescein, digoxigenin, and polyA(25) as chemical handles also successfully separated target files from the database. f , A large-scale background mimicking diverse data was created using error prone PCR 13 to mutagenize and amplify File 1. g , Random access was compared directly to chemical handle extractions. File 3 strands, with a starting fraction of 0.03% of the total number of strands, were enriched over a high-capacity background equivalent to 5.53 TB of undesired, non-specific strands using either random access (black) or PCR followed by chemical handle primer extractions (blue, green, purple or pink). After 5, 15, and 30 cycles of PCR (random access), enrichment of File 3 was 0.0%, 0.0%, and 1.69% of the total sample, respectively. After biotin-modified PCR followed by extraction, the enrichment of File 3 was 0.2%, 87.5%, and 100% of the total sample, respectively. After fluorescein-modified PCR followed by extraction, the enrichment of File 3 was 0.1%, 49.6%, and 100% of the total sample, respectively. After digoxigenin-modified PCR followed by extraction, the enrichment of File 3 was 0.2%, 14.2%, and 100% of the total sample, respectively. After poly(A)-25-modified PCR followed by extraction, the enrichment of File 3 was 0.09%, 0.47%, and 100% of the total sample, respectively.

Techniques Used: Sequencing, Next-Generation Sequencing, Polymerase Chain Reaction, Real-time Polymerase Chain Reaction, Labeling, Magnetic Beads, Modification

5) Product Images from "Genotyping 1000 yeast strains by next-generation sequencing"

Article Title: Genotyping 1000 yeast strains by next-generation sequencing

Journal: BMC Genomics

doi: 10.1186/1471-2164-14-90

Library preparation pipeline. DNA isolation is performed with a 96-head liquid handling robot. DNA fragmentation is achieved by sonication, either in glass tubes (Covaris) or PCR strips (Bandelin). SPRI bead cleanup is automated on a 96-head liquid handling robot. Three enzymatic steps for barcoded adapter ligation are performed by addition of enzyme (+ buffer), incubation, and heat inactivation in a thermocycler. After pooling of 48 barcoded libraries, samples are concentrated and size-selected using an E-gel. PCR is performed on the size-selected pool to enrich for adapter containing fragments and elongate them to full-length libraries. A final cleanup is performed in PCR strips mounted to a homemade magnetic stand.
Figure Legend Snippet: Library preparation pipeline. DNA isolation is performed with a 96-head liquid handling robot. DNA fragmentation is achieved by sonication, either in glass tubes (Covaris) or PCR strips (Bandelin). SPRI bead cleanup is automated on a 96-head liquid handling robot. Three enzymatic steps for barcoded adapter ligation are performed by addition of enzyme (+ buffer), incubation, and heat inactivation in a thermocycler. After pooling of 48 barcoded libraries, samples are concentrated and size-selected using an E-gel. PCR is performed on the size-selected pool to enrich for adapter containing fragments and elongate them to full-length libraries. A final cleanup is performed in PCR strips mounted to a homemade magnetic stand.

Techniques Used: DNA Extraction, Sonication, Polymerase Chain Reaction, Ligation, Incubation

6) Product Images from "High resolution profiling of coral-associated bacterial communities using full-length 16S rRNA sequence data from PacBio SMRT sequencing system"

Article Title: High resolution profiling of coral-associated bacterial communities using full-length 16S rRNA sequence data from PacBio SMRT sequencing system

Journal: Scientific Reports

doi: 10.1038/s41598-017-03139-4

Two-step generation of 16S amplicons for PacBio sequencing using M13-tagged barcoded primers. The first round of PCR amplifies full-length 16S rRNA fragments using gene-specific primers tailed with universal M13 forward or reverse sequence. In the second step, a unique combination of barcode sequences is added to 16S amplicons from each sample using M13 forward and reverse primers tagged with 16-base PacBio barcodes at their 5′ ends. Barcoded amplicons are subsequently pooled for SMRTbell library construction and multiplexed sequencing.
Figure Legend Snippet: Two-step generation of 16S amplicons for PacBio sequencing using M13-tagged barcoded primers. The first round of PCR amplifies full-length 16S rRNA fragments using gene-specific primers tailed with universal M13 forward or reverse sequence. In the second step, a unique combination of barcode sequences is added to 16S amplicons from each sample using M13 forward and reverse primers tagged with 16-base PacBio barcodes at their 5′ ends. Barcoded amplicons are subsequently pooled for SMRTbell library construction and multiplexed sequencing.

Techniques Used: Sequencing, Polymerase Chain Reaction

Comparison of species detection between full-length and partial 16S rRNA sequences. A neighbor-joining phylogenetic tree of 20 species identified in coral-associated microbiome generated using MEGA7. Reference type strain sequences were obtained from the RDP database. The clustering of the sequences was tested by a bootstrap approach using 1,000 replications. Orange, green and blue boxes indicate species that are classifiable based on sequence information from full-length, V3-V4 and V5-V6 regions of the 16S rRNA genes, respectively.
Figure Legend Snippet: Comparison of species detection between full-length and partial 16S rRNA sequences. A neighbor-joining phylogenetic tree of 20 species identified in coral-associated microbiome generated using MEGA7. Reference type strain sequences were obtained from the RDP database. The clustering of the sequences was tested by a bootstrap approach using 1,000 replications. Orange, green and blue boxes indicate species that are classifiable based on sequence information from full-length, V3-V4 and V5-V6 regions of the 16S rRNA genes, respectively.

Techniques Used: Generated, Sequencing

A histogram illustrating proportions of sequence reads from each sampling site that are classifiable at the species level using full-length (orange) or partial 16S rRNA gene fragments (V3-V4, green; V5-V6, blue).
Figure Legend Snippet: A histogram illustrating proportions of sequence reads from each sampling site that are classifiable at the species level using full-length (orange) or partial 16S rRNA gene fragments (V3-V4, green; V5-V6, blue).

Techniques Used: Sequencing, Sampling

7) Product Images from "Urban park soil microbiomes are a rich reservoir of natural product biosynthetic diversity"

Article Title: Urban park soil microbiomes are a rich reservoir of natural product biosynthetic diversity

Journal: Proceedings of the National Academy of Sciences of the United States of America

doi: 10.1073/pnas.1615581113

Surveying NP biosynthesis by phylogenetic analysis of PCR amplicons. ( A ) In NRP and PK biosynthesis, NPs are produced by megasynth(et)ases in thio-templated assembly line fashion. Each module in a megasynth(et)ase is composed of a highly conserved set of domains and is responsible for the incorporation of one amino acid or coenzyme A (CoA)-based building block into the growing NP. ( B ) Phylogenetic analysis of domain fragments PCR-amplified from eDNA can be used to study biosynthetic diversity hidden in soil microbiomes.
Figure Legend Snippet: Surveying NP biosynthesis by phylogenetic analysis of PCR amplicons. ( A ) In NRP and PK biosynthesis, NPs are produced by megasynth(et)ases in thio-templated assembly line fashion. Each module in a megasynth(et)ase is composed of a highly conserved set of domains and is responsible for the incorporation of one amino acid or coenzyme A (CoA)-based building block into the growing NP. ( B ) Phylogenetic analysis of domain fragments PCR-amplified from eDNA can be used to study biosynthetic diversity hidden in soil microbiomes.

Techniques Used: Polymerase Chain Reaction, Produced, Blocking Assay, Amplification

8) Product Images from "A high throughput screen for active human transposable elements"

Article Title: A high throughput screen for active human transposable elements

Journal: BMC Genomics

doi: 10.1186/s12864-018-4485-4

TE-NGS sequencing workflow. Enrichment for genomic fragments spanning active TEs and their unique flanking sequence is achieved by several enzymatic steps as described in the main text. First, genomic DNA is sheared, and adapters for sequencing are ligated to the genomic fragments following standard library preparation protocols. Next, a small aliquot (10 ng) of library is used as template for targeted amplification with primers complementary to TE subfamily-specific sequences and to the Illumina Universal PCR (P5) primer. Remaining genomic background fragments and inverted TEs in head-to-head orientation are removed by ssDNA exonuclease digestion after linear PCR amplification with TE-target primers or Illumina Universal primer, respectively. Last, amplification with nested primers targeting TE diagnostic bases, and containing Illumina i7 index and P7 primer sequences generates full double-stranded dual-adapter libraries containing unique indices for each sample and each TE subfamily, allowing for downstream pooling and multiplexing of many samples simultaneously. High throughput sequencing followed by alignment to the reference genome demarcates the TE insertion site by its 3′ end (read 2) and unique flanking sequence (read 1). TE insertions present in the reference genome can be identified by clustering of read pairs, whereas read 2 generated from polymorphic or novel TE insertions absent from the reference will map with lower quality and/or not at all; these TE can be identified by clusters of read 1 alone (see Methods; Supplemental Material for detailed procedures)
Figure Legend Snippet: TE-NGS sequencing workflow. Enrichment for genomic fragments spanning active TEs and their unique flanking sequence is achieved by several enzymatic steps as described in the main text. First, genomic DNA is sheared, and adapters for sequencing are ligated to the genomic fragments following standard library preparation protocols. Next, a small aliquot (10 ng) of library is used as template for targeted amplification with primers complementary to TE subfamily-specific sequences and to the Illumina Universal PCR (P5) primer. Remaining genomic background fragments and inverted TEs in head-to-head orientation are removed by ssDNA exonuclease digestion after linear PCR amplification with TE-target primers or Illumina Universal primer, respectively. Last, amplification with nested primers targeting TE diagnostic bases, and containing Illumina i7 index and P7 primer sequences generates full double-stranded dual-adapter libraries containing unique indices for each sample and each TE subfamily, allowing for downstream pooling and multiplexing of many samples simultaneously. High throughput sequencing followed by alignment to the reference genome demarcates the TE insertion site by its 3′ end (read 2) and unique flanking sequence (read 1). TE insertions present in the reference genome can be identified by clustering of read pairs, whereas read 2 generated from polymorphic or novel TE insertions absent from the reference will map with lower quality and/or not at all; these TE can be identified by clusters of read 1 alone (see Methods; Supplemental Material for detailed procedures)

Techniques Used: Next-Generation Sequencing, Sequencing, Amplification, Polymerase Chain Reaction, Diagnostic Assay, Multiplexing, Generated

9) Product Images from "DNA nanomapping using CRISPR-Cas9 as a programmable nanoparticle"

Article Title: DNA nanomapping using CRISPR-Cas9 as a programmable nanoparticle

Journal: Nature Communications

doi: 10.1038/s41467-017-01891-9

Detecting Cas9 labels with a DVD player part used as a displacement sensor. a Photo of the DVD player optical pickup unit showing the location of the laser-focusing objective and the signal and control connection point. Behind the objective lens is the 650-nm-wavelength diode laser, quadrant photodiode, and associated optics. b Schematic of the HS-AFM scan stage with the DVD optical pickup used as the cantilever displacement sensor. c An example image of TERT amplicons labeled with two Cas9 proteins. This image was obtained by averaging five full frames collected at the maximum frame rate of 2 frames per second. d Typical height cross section from double-labeled molecules obtained with the DVD optical pickup. e Histograms of Cas9 marker spacing measured for populations of TERT and HER2 molecules. The white asterisk indicates the expected value
Figure Legend Snippet: Detecting Cas9 labels with a DVD player part used as a displacement sensor. a Photo of the DVD player optical pickup unit showing the location of the laser-focusing objective and the signal and control connection point. Behind the objective lens is the 650-nm-wavelength diode laser, quadrant photodiode, and associated optics. b Schematic of the HS-AFM scan stage with the DVD optical pickup used as the cantilever displacement sensor. c An example image of TERT amplicons labeled with two Cas9 proteins. This image was obtained by averaging five full frames collected at the maximum frame rate of 2 frames per second. d Typical height cross section from double-labeled molecules obtained with the DVD optical pickup. e Histograms of Cas9 marker spacing measured for populations of TERT and HER2 molecules. The white asterisk indicates the expected value

Techniques Used: Labeling, Marker

sgRNA–Cas9 complexes on DNA. a Map and the corresponding HS-AFM image of Cas9-labeled BRCA1 amplicon. Red tick marks on the map indicate locations of the Alu repeat-targeted sgRNA-binding sites. In the image, sgRNA–Cas9 protein complexes appear as 3-nm-tall bumps on the DNA backbone. The amplicon sequence includes five perfect matches to the 20-bp sgRNA sequence and five single base-pair mismatches. Single base-pair mismatches label at roughly one-half the frequency of perfect matches. The star (*) indicates Cas9 bound at locations with a single mismatch, while all other locations are perfect matches. b , c Cas-9-labeled TERT and HER2 amplicons. The sharp bend induced in the DNA backbone by the sgRNA–Cas9 complex is evident in both examples
Figure Legend Snippet: sgRNA–Cas9 complexes on DNA. a Map and the corresponding HS-AFM image of Cas9-labeled BRCA1 amplicon. Red tick marks on the map indicate locations of the Alu repeat-targeted sgRNA-binding sites. In the image, sgRNA–Cas9 protein complexes appear as 3-nm-tall bumps on the DNA backbone. The amplicon sequence includes five perfect matches to the 20-bp sgRNA sequence and five single base-pair mismatches. Single base-pair mismatches label at roughly one-half the frequency of perfect matches. The star (*) indicates Cas9 bound at locations with a single mismatch, while all other locations are perfect matches. b , c Cas-9-labeled TERT and HER2 amplicons. The sharp bend induced in the DNA backbone by the sgRNA–Cas9 complex is evident in both examples

Techniques Used: Labeling, Amplification, Binding Assay, Sequencing

Mapping BCL2–IGH translocations with Cas9. a Maps of PCR amplicons from wild-type BCL2 and IGH with Cas9 labels (different colors for emphasis). The two labels on BCL2 straddle the 300-bp MBR translocation hotspot. b HSAFM images of labeled amplicons. c HSAFM-determined maps of the translocation breakpoints from a follicular lymphoma cell line DOHH2, and the seven patient samples. Identity of the Cas9 labels is determined by measuring their position from the end of the molecule. Maps were confirmed with 10 or more molecules each. In all seven patent samples, the translocation breakpoint mapped to the major breakpoint region (MBR) of BCL2 and the J H region of IGH
Figure Legend Snippet: Mapping BCL2–IGH translocations with Cas9. a Maps of PCR amplicons from wild-type BCL2 and IGH with Cas9 labels (different colors for emphasis). The two labels on BCL2 straddle the 300-bp MBR translocation hotspot. b HSAFM images of labeled amplicons. c HSAFM-determined maps of the translocation breakpoints from a follicular lymphoma cell line DOHH2, and the seven patient samples. Identity of the Cas9 labels is determined by measuring their position from the end of the molecule. Maps were confirmed with 10 or more molecules each. In all seven patent samples, the translocation breakpoint mapped to the major breakpoint region (MBR) of BCL2 and the J H region of IGH

Techniques Used: Polymerase Chain Reaction, Translocation Assay, Labeling

10) Product Images from "Culture-independent discovery of the malacidins as calcium-dependent antibiotics with activity against multidrug-resistant Gram-positive pathogens"

Article Title: Culture-independent discovery of the malacidins as calcium-dependent antibiotics with activity against multidrug-resistant Gram-positive pathogens

Journal: Nature microbiology

doi: 10.1038/s41564-018-0110-1

Using a culture-independent strategy for the discovery of calcium-dependent antibiotics from the global microbiome (a) i ) Degenerate PCR primers targeting the conserved regions of adenylation domains (AD) found in nonribosomal peptide synthetase genes were used to generate amplicons from an arrayed collection of environmental DNA isolated from 2000 unique soils. The reads from these next-generation sequenced amplicons (natural product sequence tags, NPSTs) were analyzed by eSNaPD (environmental Surveyor of Natural Product Diversity). ii) A desert soil rich in AD-NPSTs from the previously unknown malacidin clade was used to build an arrayed cosmid library. Cosmids harboring all fragments of a targeted biosynthetic gene cluster were assembled and integrated into a heterologous host for production, extraction, and characterization. (b) AD-NPSTs identified by the eSNaPD analysis to be evolutionarily related to the conserved Asp4 ADs of known calcium-dependent antibiotics were used to phylogenetically map the unexplored clades of this larger family across all tested soil microbiomes. The subfamilies of calcium-dependent antibiotics and their relative abundance are illustrated on the phylogenetic tree by color and percent. Across all sampled soil metagenomes, the malacidin antibiotic-clade represents 19% of the NPSTs, and 59% of calcium-dependent antibiotic tags originate from unexplored branches. (c) Geospatial distribution of calcium-dependent antibiotics across sampled US soil metagenomes. US states containing at least one soil with AD-NPSTs from the malacidin clade are indicated in orange. States lacking malacidin tags but still containing calcium-dependent antibiotics NPSTs are indicated in blue. States with at least one sampled soil but no detected calcium-dependent antibiotics NPSTs are highlighted in dark grey.
Figure Legend Snippet: Using a culture-independent strategy for the discovery of calcium-dependent antibiotics from the global microbiome (a) i ) Degenerate PCR primers targeting the conserved regions of adenylation domains (AD) found in nonribosomal peptide synthetase genes were used to generate amplicons from an arrayed collection of environmental DNA isolated from 2000 unique soils. The reads from these next-generation sequenced amplicons (natural product sequence tags, NPSTs) were analyzed by eSNaPD (environmental Surveyor of Natural Product Diversity). ii) A desert soil rich in AD-NPSTs from the previously unknown malacidin clade was used to build an arrayed cosmid library. Cosmids harboring all fragments of a targeted biosynthetic gene cluster were assembled and integrated into a heterologous host for production, extraction, and characterization. (b) AD-NPSTs identified by the eSNaPD analysis to be evolutionarily related to the conserved Asp4 ADs of known calcium-dependent antibiotics were used to phylogenetically map the unexplored clades of this larger family across all tested soil microbiomes. The subfamilies of calcium-dependent antibiotics and their relative abundance are illustrated on the phylogenetic tree by color and percent. Across all sampled soil metagenomes, the malacidin antibiotic-clade represents 19% of the NPSTs, and 59% of calcium-dependent antibiotic tags originate from unexplored branches. (c) Geospatial distribution of calcium-dependent antibiotics across sampled US soil metagenomes. US states containing at least one soil with AD-NPSTs from the malacidin clade are indicated in orange. States lacking malacidin tags but still containing calcium-dependent antibiotics NPSTs are indicated in blue. States with at least one sampled soil but no detected calcium-dependent antibiotics NPSTs are highlighted in dark grey.

Techniques Used: Polymerase Chain Reaction, Isolation, Sequencing

11) Product Images from "Genotyping 1000 yeast strains by next-generation sequencing"

Article Title: Genotyping 1000 yeast strains by next-generation sequencing

Journal: BMC Genomics

doi: 10.1186/1471-2164-14-90

Quality control of fragmented DNA. ( A ) Bioanalyzer results from three DNA samples fragmented either in glass tubes with a Covaris DNA shearing device (Duty cycle 10%, Intensity 4.5, Cycles per burst 200, Time 120 s), or in PCR strips with a Bandelin sonicator (2 times 4 min). ( B ) 1.5% agarose gel loaded with 22 samples fragmented by Bandelin sonication. The size distribution is very narrow (major peak between 100–300 bp) and has an acceptable reproducibility.
Figure Legend Snippet: Quality control of fragmented DNA. ( A ) Bioanalyzer results from three DNA samples fragmented either in glass tubes with a Covaris DNA shearing device (Duty cycle 10%, Intensity 4.5, Cycles per burst 200, Time 120 s), or in PCR strips with a Bandelin sonicator (2 times 4 min). ( B ) 1.5% agarose gel loaded with 22 samples fragmented by Bandelin sonication. The size distribution is very narrow (major peak between 100–300 bp) and has an acceptable reproducibility.

Techniques Used: Polymerase Chain Reaction, Agarose Gel Electrophoresis, Sonication

Library preparation pipeline. DNA isolation is performed with a 96-head liquid handling robot. DNA fragmentation is achieved by sonication, either in glass tubes (Covaris) or PCR strips (Bandelin). SPRI bead cleanup is automated on a 96-head liquid handling robot. Three enzymatic steps for barcoded adapter ligation are performed by addition of enzyme (+ buffer), incubation, and heat inactivation in a thermocycler. After pooling of 48 barcoded libraries, samples are concentrated and size-selected using an E-gel. PCR is performed on the size-selected pool to enrich for adapter containing fragments and elongate them to full-length libraries. A final cleanup is performed in PCR strips mounted to a homemade magnetic stand.
Figure Legend Snippet: Library preparation pipeline. DNA isolation is performed with a 96-head liquid handling robot. DNA fragmentation is achieved by sonication, either in glass tubes (Covaris) or PCR strips (Bandelin). SPRI bead cleanup is automated on a 96-head liquid handling robot. Three enzymatic steps for barcoded adapter ligation are performed by addition of enzyme (+ buffer), incubation, and heat inactivation in a thermocycler. After pooling of 48 barcoded libraries, samples are concentrated and size-selected using an E-gel. PCR is performed on the size-selected pool to enrich for adapter containing fragments and elongate them to full-length libraries. A final cleanup is performed in PCR strips mounted to a homemade magnetic stand.

Techniques Used: DNA Extraction, Sonication, Polymerase Chain Reaction, Ligation, Incubation

12) Product Images from "Preclinical Characterization of GSK2336805, a Novel Inhibitor of Hepatitis C Virus Replication That Selects for Resistance in NS5A"

Article Title: Preclinical Characterization of GSK2336805, a Novel Inhibitor of Hepatitis C Virus Replication That Selects for Resistance in NS5A

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.01363-13

Activity of GSK2336805 on chimeric replicons containing genotype 1a and 1b NS5A patient sequences. GSK2336805 was tested in transient-transfection assays for activity on chimeric replicons containing genotype 1a or 1b patient sequences. The wild-type replicon is the genotype 1b Con-1 ET replicon, the backbone for the chimeric replicons, represented by the open circles. EC 50 s for each chimeric replicon are represented by black circles. The solid lines represent the mean EC 50 for the chimeric replicons, and the dashed lines are the 95% confidence intervals for GSK2336805 activity. Each replicon was tested at least 3 times.
Figure Legend Snippet: Activity of GSK2336805 on chimeric replicons containing genotype 1a and 1b NS5A patient sequences. GSK2336805 was tested in transient-transfection assays for activity on chimeric replicons containing genotype 1a or 1b patient sequences. The wild-type replicon is the genotype 1b Con-1 ET replicon, the backbone for the chimeric replicons, represented by the open circles. EC 50 s for each chimeric replicon are represented by black circles. The solid lines represent the mean EC 50 for the chimeric replicons, and the dashed lines are the 95% confidence intervals for GSK2336805 activity. Each replicon was tested at least 3 times.

Techniques Used: Activity Assay, Transfection

). Replicons containing the amino acid changes were tested in transient-transfection assays. The average EC 50 s are plotted on a log 10 scale, and the error bars represent the 95% confidence intervals. Each replicon was tested at least 3 times.
Figure Legend Snippet: ). Replicons containing the amino acid changes were tested in transient-transfection assays. The average EC 50 s are plotted on a log 10 scale, and the error bars represent the 95% confidence intervals. Each replicon was tested at least 3 times.

Techniques Used: Transfection

13) Product Images from "Guiding the humoral response against HIV-1 toward a MPER adjacent region by immunization with a VLP-formulated antibody-selected envelope variant"

Article Title: Guiding the humoral response against HIV-1 toward a MPER adjacent region by immunization with a VLP-formulated antibody-selected envelope variant

Journal: PLoS ONE

doi: 10.1371/journal.pone.0208345

Positional diversity detected by deep-sequencing in parental virus AC10_29 (A) and the mutant library before [L; (B)] and after selection by the R.I.S. method [4E10sL; (C)]. (A) Amplicon sequencing of the original primary isolate before random mutagenenesis of the env gene revealed a homogeneous population. (B) The library generated by random mutagenesis had a wide variability of mutations across the amplicons sequenced. (C) Some mutations are fixed after selection with 4E10 by the R.I.S. method. Positional diversity is calculated as the frequency of all nucleotides that differ from the parental strain (AC10_29) at each evaluated position. Insertions and deletions are taken into account. Genomic regions corresponding to each amplicon are indicated.
Figure Legend Snippet: Positional diversity detected by deep-sequencing in parental virus AC10_29 (A) and the mutant library before [L; (B)] and after selection by the R.I.S. method [4E10sL; (C)]. (A) Amplicon sequencing of the original primary isolate before random mutagenenesis of the env gene revealed a homogeneous population. (B) The library generated by random mutagenesis had a wide variability of mutations across the amplicons sequenced. (C) Some mutations are fixed after selection with 4E10 by the R.I.S. method. Positional diversity is calculated as the frequency of all nucleotides that differ from the parental strain (AC10_29) at each evaluated position. Insertions and deletions are taken into account. Genomic regions corresponding to each amplicon are indicated.

Techniques Used: Sequencing, Mutagenesis, Selection, Amplification, Generated

14) Product Images from "Diversity and functions of the sheep faecal microbiota: a multi‐omic characterization"

Article Title: Diversity and functions of the sheep faecal microbiota: a multi‐omic characterization

Journal: Microbial Biotechnology

doi: 10.1111/1751-7915.12462

Taxonomic composition of the sheep faecal prokaryotic microbiota, according to V4‐16S rRNA (V4‐ MG , left), metagenomic (S‐ MG , centre) and metaproteomic ( MP , right) results. (A) Tukey's box plot illustrating the microbiota composition at phylum level. The top 10 phyla are shown, ordered by decreasing mean relative abundance. (B) Tukey's box plot illustrating the microbiota composition at family level. The top 20 families are shown, grouped based on the relative phylum (A, Actinobacteria; B, Bacteroidetes; E, Euryarchaeota; F, Firmicutes; FB , Fibrobacteres; P, Proteobacteria; S, Spirochaetes; V, Verrucomicrobia) and further ordered by decreasing mean relative abundance.
Figure Legend Snippet: Taxonomic composition of the sheep faecal prokaryotic microbiota, according to V4‐16S rRNA (V4‐ MG , left), metagenomic (S‐ MG , centre) and metaproteomic ( MP , right) results. (A) Tukey's box plot illustrating the microbiota composition at phylum level. The top 10 phyla are shown, ordered by decreasing mean relative abundance. (B) Tukey's box plot illustrating the microbiota composition at family level. The top 20 families are shown, grouped based on the relative phylum (A, Actinobacteria; B, Bacteroidetes; E, Euryarchaeota; F, Firmicutes; FB , Fibrobacteres; P, Proteobacteria; S, Spirochaetes; V, Verrucomicrobia) and further ordered by decreasing mean relative abundance.

Techniques Used:

15) Product Images from "Untangling Species-Level Composition of Complex Bacterial Communities through a Novel Metagenomic Approach"

Article Title: Untangling Species-Level Composition of Complex Bacterial Communities through a Novel Metagenomic Approach

Journal: mSystems

doi: 10.1128/mSystems.00404-20

In silico evaluation of microbial ITS profiling performance and comparison respect to different hypervariable 16S rRNA gene regions. (a) Percentage of bacterial taxa correctly classified by ITS and 16S rRNA gene profiling methods with respect to expected taxonomic profiles of artificial data sets. (b) Taxonomic profiles observed using ITS and 16S rRNA gene profiling methods and their comparison with respect to expected taxonomic profiles of artificial data sets.
Figure Legend Snippet: In silico evaluation of microbial ITS profiling performance and comparison respect to different hypervariable 16S rRNA gene regions. (a) Percentage of bacterial taxa correctly classified by ITS and 16S rRNA gene profiling methods with respect to expected taxonomic profiles of artificial data sets. (b) Taxonomic profiles observed using ITS and 16S rRNA gene profiling methods and their comparison with respect to expected taxonomic profiles of artificial data sets.

Techniques Used: In Silico

Performance of microbial ITS profiling compared to 16S rRNA gene cataloguing and read-based classification of shotgun metagenomic data sets. (a) Average percentage of reads classified to the species level using ITS and 16S rRNA gene profiling for each matrix analyzed. Data obtained for each sample are reported in Data Set S4 . (b) Average percentage of reads classified by 16S rRNA gene profiling as a genus also detected by microbial ITS profiling of the same read, for each matrix analyzed. Data obtained for each sample are reported in Data Set S4 . (c) Percentage of reads classified at the species level based on read length.
Figure Legend Snippet: Performance of microbial ITS profiling compared to 16S rRNA gene cataloguing and read-based classification of shotgun metagenomic data sets. (a) Average percentage of reads classified to the species level using ITS and 16S rRNA gene profiling for each matrix analyzed. Data obtained for each sample are reported in Data Set S4 . (b) Average percentage of reads classified by 16S rRNA gene profiling as a genus also detected by microbial ITS profiling of the same read, for each matrix analyzed. Data obtained for each sample are reported in Data Set S4 . (c) Percentage of reads classified at the species level based on read length.

Techniques Used:

16) Product Images from "Ligation-anchored PCR unveils immune repertoire of TCR-beta from whole blood"

Article Title: Ligation-anchored PCR unveils immune repertoire of TCR-beta from whole blood

Journal: BMC Biotechnology

doi: 10.1186/s12896-015-0153-9

PCR method utilizing single target-specific primer to explore actively transcribed immune genes. A) , The scheme of an integrative approach to explore immune repertoire from whole blood (P-U represents a universal primer sitting on the linker, whereas P-VDJ represents a TRB-specific primer targeting the consensus sequence in the constant region right after J-segment); B) , DNA gel image of PCR amplified variable region (V-region) of TRB gene from a healthy donor under different conditions; C) , DNA gel image of amplicons from malignant and benign patients under linker ligation or control condition.
Figure Legend Snippet: PCR method utilizing single target-specific primer to explore actively transcribed immune genes. A) , The scheme of an integrative approach to explore immune repertoire from whole blood (P-U represents a universal primer sitting on the linker, whereas P-VDJ represents a TRB-specific primer targeting the consensus sequence in the constant region right after J-segment); B) , DNA gel image of PCR amplified variable region (V-region) of TRB gene from a healthy donor under different conditions; C) , DNA gel image of amplicons from malignant and benign patients under linker ligation or control condition.

Techniques Used: Polymerase Chain Reaction, Sequencing, Amplification, Ligation

17) Product Images from "Culture-independent discovery of the malacidins as calcium-dependent antibiotics with activity against multidrug-resistant Gram-positive pathogens"

Article Title: Culture-independent discovery of the malacidins as calcium-dependent antibiotics with activity against multidrug-resistant Gram-positive pathogens

Journal: Nature microbiology

doi: 10.1038/s41564-018-0110-1

Using a culture-independent strategy for the discovery of calcium-dependent antibiotics from the global microbiome (a) i ) Degenerate PCR primers targeting the conserved regions of adenylation domains (AD) found in nonribosomal peptide synthetase genes were used to generate amplicons from an arrayed collection of environmental DNA isolated from 2000 unique soils. The reads from these next-generation sequenced amplicons (natural product sequence tags, NPSTs) were analyzed by eSNaPD (environmental Surveyor of Natural Product Diversity). ii) A desert soil rich in AD-NPSTs from the previously unknown malacidin clade was used to build an arrayed cosmid library. Cosmids harboring all fragments of a targeted biosynthetic gene cluster were assembled and integrated into a heterologous host for production, extraction, and characterization. (b) AD-NPSTs identified by the eSNaPD analysis to be evolutionarily related to the conserved Asp4 ADs of known calcium-dependent antibiotics were used to phylogenetically map the unexplored clades of this larger family across all tested soil microbiomes. The subfamilies of calcium-dependent antibiotics and their relative abundance are illustrated on the phylogenetic tree by color and percent. Across all sampled soil metagenomes, the malacidin antibiotic-clade represents 19% of the NPSTs, and 59% of calcium-dependent antibiotic tags originate from unexplored branches. (c) Geospatial distribution of calcium-dependent antibiotics across sampled US soil metagenomes. US states containing at least one soil with AD-NPSTs from the malacidin clade are indicated in orange. States lacking malacidin tags but still containing calcium-dependent antibiotics NPSTs are indicated in blue. States with at least one sampled soil but no detected calcium-dependent antibiotics NPSTs are highlighted in dark grey.
Figure Legend Snippet: Using a culture-independent strategy for the discovery of calcium-dependent antibiotics from the global microbiome (a) i ) Degenerate PCR primers targeting the conserved regions of adenylation domains (AD) found in nonribosomal peptide synthetase genes were used to generate amplicons from an arrayed collection of environmental DNA isolated from 2000 unique soils. The reads from these next-generation sequenced amplicons (natural product sequence tags, NPSTs) were analyzed by eSNaPD (environmental Surveyor of Natural Product Diversity). ii) A desert soil rich in AD-NPSTs from the previously unknown malacidin clade was used to build an arrayed cosmid library. Cosmids harboring all fragments of a targeted biosynthetic gene cluster were assembled and integrated into a heterologous host for production, extraction, and characterization. (b) AD-NPSTs identified by the eSNaPD analysis to be evolutionarily related to the conserved Asp4 ADs of known calcium-dependent antibiotics were used to phylogenetically map the unexplored clades of this larger family across all tested soil microbiomes. The subfamilies of calcium-dependent antibiotics and their relative abundance are illustrated on the phylogenetic tree by color and percent. Across all sampled soil metagenomes, the malacidin antibiotic-clade represents 19% of the NPSTs, and 59% of calcium-dependent antibiotic tags originate from unexplored branches. (c) Geospatial distribution of calcium-dependent antibiotics across sampled US soil metagenomes. US states containing at least one soil with AD-NPSTs from the malacidin clade are indicated in orange. States lacking malacidin tags but still containing calcium-dependent antibiotics NPSTs are indicated in blue. States with at least one sampled soil but no detected calcium-dependent antibiotics NPSTs are highlighted in dark grey.

Techniques Used: Polymerase Chain Reaction, Isolation, Sequencing

18) Product Images from "DNA nanomapping using CRISPR-Cas9 as a programmable nanoparticle"

Article Title: DNA nanomapping using CRISPR-Cas9 as a programmable nanoparticle

Journal: Nature Communications

doi: 10.1038/s41467-017-01891-9

Detecting Cas9 labels with a DVD player part used as a displacement sensor. a Photo of the DVD player optical pickup unit showing the location of the laser-focusing objective and the signal and control connection point. Behind the objective lens is the 650-nm-wavelength diode laser, quadrant photodiode, and associated optics. b Schematic of the HS-AFM scan stage with the DVD optical pickup used as the cantilever displacement sensor. c An example image of TERT amplicons labeled with two Cas9 proteins. This image was obtained by averaging five full frames collected at the maximum frame rate of 2 frames per second. d Typical height cross section from double-labeled molecules obtained with the DVD optical pickup. e Histograms of Cas9 marker spacing measured for populations of TERT and HER2 molecules. The white asterisk indicates the expected value
Figure Legend Snippet: Detecting Cas9 labels with a DVD player part used as a displacement sensor. a Photo of the DVD player optical pickup unit showing the location of the laser-focusing objective and the signal and control connection point. Behind the objective lens is the 650-nm-wavelength diode laser, quadrant photodiode, and associated optics. b Schematic of the HS-AFM scan stage with the DVD optical pickup used as the cantilever displacement sensor. c An example image of TERT amplicons labeled with two Cas9 proteins. This image was obtained by averaging five full frames collected at the maximum frame rate of 2 frames per second. d Typical height cross section from double-labeled molecules obtained with the DVD optical pickup. e Histograms of Cas9 marker spacing measured for populations of TERT and HER2 molecules. The white asterisk indicates the expected value

Techniques Used: Labeling, Marker

sgRNA–Cas9 complexes on DNA. a Map and the corresponding HS-AFM image of Cas9-labeled BRCA1 amplicon. Red tick marks on the map indicate locations of the Alu repeat-targeted sgRNA-binding sites. In the image, sgRNA–Cas9 protein complexes appear as 3-nm-tall bumps on the DNA backbone. The amplicon sequence includes five perfect matches to the 20-bp sgRNA sequence and five single base-pair mismatches. Single base-pair mismatches label at roughly one-half the frequency of perfect matches. The star (*) indicates Cas9 bound at locations with a single mismatch, while all other locations are perfect matches. b , c Cas-9-labeled TERT and HER2 amplicons. The sharp bend induced in the DNA backbone by the sgRNA–Cas9 complex is evident in both examples
Figure Legend Snippet: sgRNA–Cas9 complexes on DNA. a Map and the corresponding HS-AFM image of Cas9-labeled BRCA1 amplicon. Red tick marks on the map indicate locations of the Alu repeat-targeted sgRNA-binding sites. In the image, sgRNA–Cas9 protein complexes appear as 3-nm-tall bumps on the DNA backbone. The amplicon sequence includes five perfect matches to the 20-bp sgRNA sequence and five single base-pair mismatches. Single base-pair mismatches label at roughly one-half the frequency of perfect matches. The star (*) indicates Cas9 bound at locations with a single mismatch, while all other locations are perfect matches. b , c Cas-9-labeled TERT and HER2 amplicons. The sharp bend induced in the DNA backbone by the sgRNA–Cas9 complex is evident in both examples

Techniques Used: Labeling, Amplification, Binding Assay, Sequencing

Mapping BCL2–IGH translocations with Cas9. a Maps of PCR amplicons from wild-type BCL2 and IGH with Cas9 labels (different colors for emphasis). The two labels on BCL2 straddle the 300-bp MBR translocation hotspot. b HSAFM images of labeled amplicons. c HSAFM-determined maps of the translocation breakpoints from a follicular lymphoma cell line DOHH2, and the seven patient samples. Identity of the Cas9 labels is determined by measuring their position from the end of the molecule. Maps were confirmed with 10 or more molecules each. In all seven patent samples, the translocation breakpoint mapped to the major breakpoint region (MBR) of BCL2 and the J H region of IGH
Figure Legend Snippet: Mapping BCL2–IGH translocations with Cas9. a Maps of PCR amplicons from wild-type BCL2 and IGH with Cas9 labels (different colors for emphasis). The two labels on BCL2 straddle the 300-bp MBR translocation hotspot. b HSAFM images of labeled amplicons. c HSAFM-determined maps of the translocation breakpoints from a follicular lymphoma cell line DOHH2, and the seven patient samples. Identity of the Cas9 labels is determined by measuring their position from the end of the molecule. Maps were confirmed with 10 or more molecules each. In all seven patent samples, the translocation breakpoint mapped to the major breakpoint region (MBR) of BCL2 and the J H region of IGH

Techniques Used: Polymerase Chain Reaction, Translocation Assay, Labeling

19) Product Images from "DNA nanomapping using CRISPR-Cas9 as a programmable nanoparticle"

Article Title: DNA nanomapping using CRISPR-Cas9 as a programmable nanoparticle

Journal: Nature Communications

doi: 10.1038/s41467-017-01891-9

Detecting Cas9 labels with a DVD player part used as a displacement sensor. a Photo of the DVD player optical pickup unit showing the location of the laser-focusing objective and the signal and control connection point. Behind the objective lens is the 650-nm-wavelength diode laser, quadrant photodiode, and associated optics. b Schematic of the HS-AFM scan stage with the DVD optical pickup used as the cantilever displacement sensor. c An example image of TERT amplicons labeled with two Cas9 proteins. This image was obtained by averaging five full frames collected at the maximum frame rate of 2 frames per second. d Typical height cross section from double-labeled molecules obtained with the DVD optical pickup. e Histograms of Cas9 marker spacing measured for populations of TERT and HER2 molecules. The white asterisk indicates the expected value
Figure Legend Snippet: Detecting Cas9 labels with a DVD player part used as a displacement sensor. a Photo of the DVD player optical pickup unit showing the location of the laser-focusing objective and the signal and control connection point. Behind the objective lens is the 650-nm-wavelength diode laser, quadrant photodiode, and associated optics. b Schematic of the HS-AFM scan stage with the DVD optical pickup used as the cantilever displacement sensor. c An example image of TERT amplicons labeled with two Cas9 proteins. This image was obtained by averaging five full frames collected at the maximum frame rate of 2 frames per second. d Typical height cross section from double-labeled molecules obtained with the DVD optical pickup. e Histograms of Cas9 marker spacing measured for populations of TERT and HER2 molecules. The white asterisk indicates the expected value

Techniques Used: Labeling, Marker

sgRNA–Cas9 complexes on DNA. a Map and the corresponding HS-AFM image of Cas9-labeled BRCA1 amplicon. Red tick marks on the map indicate locations of the Alu repeat-targeted sgRNA-binding sites. In the image, sgRNA–Cas9 protein complexes appear as 3-nm-tall bumps on the DNA backbone. The amplicon sequence includes five perfect matches to the 20-bp sgRNA sequence and five single base-pair mismatches. Single base-pair mismatches label at roughly one-half the frequency of perfect matches. The star (*) indicates Cas9 bound at locations with a single mismatch, while all other locations are perfect matches. b , c Cas-9-labeled TERT and HER2 amplicons. The sharp bend induced in the DNA backbone by the sgRNA–Cas9 complex is evident in both examples
Figure Legend Snippet: sgRNA–Cas9 complexes on DNA. a Map and the corresponding HS-AFM image of Cas9-labeled BRCA1 amplicon. Red tick marks on the map indicate locations of the Alu repeat-targeted sgRNA-binding sites. In the image, sgRNA–Cas9 protein complexes appear as 3-nm-tall bumps on the DNA backbone. The amplicon sequence includes five perfect matches to the 20-bp sgRNA sequence and five single base-pair mismatches. Single base-pair mismatches label at roughly one-half the frequency of perfect matches. The star (*) indicates Cas9 bound at locations with a single mismatch, while all other locations are perfect matches. b , c Cas-9-labeled TERT and HER2 amplicons. The sharp bend induced in the DNA backbone by the sgRNA–Cas9 complex is evident in both examples

Techniques Used: Labeling, Amplification, Binding Assay, Sequencing

Mapping BCL2–IGH translocations with Cas9. a Maps of PCR amplicons from wild-type BCL2 and IGH with Cas9 labels (different colors for emphasis). The two labels on BCL2 straddle the 300-bp MBR translocation hotspot. b HSAFM images of labeled amplicons. c HSAFM-determined maps of the translocation breakpoints from a follicular lymphoma cell line DOHH2, and the seven patient samples. Identity of the Cas9 labels is determined by measuring their position from the end of the molecule. Maps were confirmed with 10 or more molecules each. In all seven patent samples, the translocation breakpoint mapped to the major breakpoint region (MBR) of BCL2 and the J H region of IGH
Figure Legend Snippet: Mapping BCL2–IGH translocations with Cas9. a Maps of PCR amplicons from wild-type BCL2 and IGH with Cas9 labels (different colors for emphasis). The two labels on BCL2 straddle the 300-bp MBR translocation hotspot. b HSAFM images of labeled amplicons. c HSAFM-determined maps of the translocation breakpoints from a follicular lymphoma cell line DOHH2, and the seven patient samples. Identity of the Cas9 labels is determined by measuring their position from the end of the molecule. Maps were confirmed with 10 or more molecules each. In all seven patent samples, the translocation breakpoint mapped to the major breakpoint region (MBR) of BCL2 and the J H region of IGH

Techniques Used: Polymerase Chain Reaction, Translocation Assay, Labeling

20) Product Images from "Mycobiome: Approaches to Analysis of Intestinal Fungi"

Article Title: Mycobiome: Approaches to Analysis of Intestinal Fungi

Journal: Journal of immunological methods

doi: 10.1016/j.jim.2015.04.004

Fragmentation of ITS amplicons before sequencing decreases the bias during PGM sequencing
Figure Legend Snippet: Fragmentation of ITS amplicons before sequencing decreases the bias during PGM sequencing

Techniques Used: Sequencing

Fungal ITS amplicons from different species vary in size and content while bacterial 16S amplicons are similar
Figure Legend Snippet: Fungal ITS amplicons from different species vary in size and content while bacterial 16S amplicons are similar

Techniques Used:

21) Product Images from "The Detailed 3D Multi-Loop Aggregate/Rosette Chromatin Architecture and Functional Dynamic Organization of the Human and Mouse Genomes"

Article Title: The Detailed 3D Multi-Loop Aggregate/Rosette Chromatin Architecture and Functional Dynamic Organization of the Human and Mouse Genomes

Journal: bioRxiv

doi: 10.1101/064642

T2C description, interaction mapping, and direct determination of the chromatin quasi-fibre and the aggregated loop/rosette 3D architecture of the human and mouse genomes: A , Cell nuclei in a population of cells (transmission light and fluorescence microscopy, Fejes-Toth et al., 2004 ) have an underlying chromatin architecture (simulated cell nucleus containing 1.2 million polymer segments; resolution 5.2 kbp, i.e. ∼50 nucleosomes; Multi-Loop-Subcompartment (MLS) rosette model with 126 kbp loops and linkers; Knoch, 2002 ). After crosslinking the DNA is restricted within the nucleus by a 1st restriction enzyme, before the cross-linked fragments are extracted and diluted such that intra fragment re-ligation is favoured. After de-crosslinking, the re-ligated material is shortened by a 2nd restriction enzyme or sonication and purified by a capture array with oligos designed next to the 1st restriction enzyme, before paired-end-sequencing over the ligation position. After alignment to the reference genome, this results in interactions frequency matrices (B-D) and scaling curves ( Fig. 2 ). B, C , Interaction matrices (logarithmic and colour coded scale; left right) of the human IGF/H19 11p 15.5-15.4 region (B) in HB2, HEK293T TEV (intact cohesin) and HEK293T HRV (cleaved cohesin) as well as the mouse β-g 7qE3-F1 region (C) for fetal brain (inactive β-globin) and liver cells (active β-globin) show the formation of subchromosomal domains separated by a linker (borders: pink lines, right; D1s, D1e: start and end of domains), which consist of loops (red lines; 8L: number of loops), representing due to the grid-like pattern loop aggregates/rosettes. A grid-like pattern is also visible in the interactions between the domains and correspond to the interactions of loops and loop bases of interacting domains. Near the diagonal the aggregation into a chromatin quasi-fibre as well as loop internal structures are visible (zooming in- and out the images can make this clearer). Between different cell types or functional states only some local differences are visible resulting in a consensus architecture and allowing simulation of the 3D architecture (middle; resolution
Figure Legend Snippet: T2C description, interaction mapping, and direct determination of the chromatin quasi-fibre and the aggregated loop/rosette 3D architecture of the human and mouse genomes: A , Cell nuclei in a population of cells (transmission light and fluorescence microscopy, Fejes-Toth et al., 2004 ) have an underlying chromatin architecture (simulated cell nucleus containing 1.2 million polymer segments; resolution 5.2 kbp, i.e. ∼50 nucleosomes; Multi-Loop-Subcompartment (MLS) rosette model with 126 kbp loops and linkers; Knoch, 2002 ). After crosslinking the DNA is restricted within the nucleus by a 1st restriction enzyme, before the cross-linked fragments are extracted and diluted such that intra fragment re-ligation is favoured. After de-crosslinking, the re-ligated material is shortened by a 2nd restriction enzyme or sonication and purified by a capture array with oligos designed next to the 1st restriction enzyme, before paired-end-sequencing over the ligation position. After alignment to the reference genome, this results in interactions frequency matrices (B-D) and scaling curves ( Fig. 2 ). B, C , Interaction matrices (logarithmic and colour coded scale; left right) of the human IGF/H19 11p 15.5-15.4 region (B) in HB2, HEK293T TEV (intact cohesin) and HEK293T HRV (cleaved cohesin) as well as the mouse β-g 7qE3-F1 region (C) for fetal brain (inactive β-globin) and liver cells (active β-globin) show the formation of subchromosomal domains separated by a linker (borders: pink lines, right; D1s, D1e: start and end of domains), which consist of loops (red lines; 8L: number of loops), representing due to the grid-like pattern loop aggregates/rosettes. A grid-like pattern is also visible in the interactions between the domains and correspond to the interactions of loops and loop bases of interacting domains. Near the diagonal the aggregation into a chromatin quasi-fibre as well as loop internal structures are visible (zooming in- and out the images can make this clearer). Between different cell types or functional states only some local differences are visible resulting in a consensus architecture and allowing simulation of the 3D architecture (middle; resolution

Techniques Used: Transmission Assay, Fluorescence, Microscopy, Ligation, Sonication, Purification, Sequencing, Functional Assay

Scaling analysis of experiments, simulations, and the DNA sequence showing the formation of a chromatin quasi-fibre and the loop aggregate/rosette genome architecture: A , The fine-structured multi-scaling resulting from the T2C interaction frequency as a function of the genomic separation for the human IGF/H19 11p 15.5-15.4 region and the mouse β-globin locus 7qE3-F1 (3 bp average (1-200 bp) and thereafter a grouping with a 1% resolution per order of magnitude which for clarity is smoothed by a running window average for > 10 3 bp; see also Fig. S8; the values
Figure Legend Snippet: Scaling analysis of experiments, simulations, and the DNA sequence showing the formation of a chromatin quasi-fibre and the loop aggregate/rosette genome architecture: A , The fine-structured multi-scaling resulting from the T2C interaction frequency as a function of the genomic separation for the human IGF/H19 11p 15.5-15.4 region and the mouse β-globin locus 7qE3-F1 (3 bp average (1-200 bp) and thereafter a grouping with a 1% resolution per order of magnitude which for clarity is smoothed by a running window average for > 10 3 bp; see also Fig. S8; the values

Techniques Used: Sequencing

22) Product Images from "Loss of compensatory pro-survival and anti-apoptotic modulator, IKKε, sensitizes ovarian cancer cells to CHEK1 loss through an increased level of p21"

Article Title: Loss of compensatory pro-survival and anti-apoptotic modulator, IKKε, sensitizes ovarian cancer cells to CHEK1 loss through an increased level of p21

Journal: Oncotarget

doi:

Validation of CHEK1 lethal effects in IKKε-mtached Ovcar5 cells (A) The knockdown levels of CHEK1 by 3 different shRNAs were examined by Western blot analysis. GAPDH was used as a loading control. (B) Double-positive cells after IKKε and CHEK1 shRNA knockdown were monitored by flow cytometry after two rounds of infection. Dot plots at 7 doubling times were shown. IKKε shRNA constructs co-express LYT2 (mouse CD8α) which was detected by an antibody conjugated to APC; CHEK1 shRNA constructs co-expressed GFP. (C) Two previously validated IKKε shRNAs were used. Either IKKε #1 or IKKε #2 shRNA (in pRSM-LYT2 vector) was co-infected with one of 3 different CHEK1 shRNAs (in pRSMX-PG vector) into Ovcar5 cells. The changes in double-positive cells were measured and calculated relative to 4 doublings after infection, at which GFP signal is maximal. The average number from 3 different CHEK1 shRNA in either negative shRNA or IKKε shRNA were calculated during indicated time courses. The significant p value was calculated by a 2-sided student's t-test. (D) A2780 IKKε-matched cell lines were established and shRNA library screening performed in the same manner as in Ovcar5. Using a cut-off with a fold change of ≤ 0.7 with a p -value of ≤ 0.05 shown in bold, 3 different CHEK1 shRNAs were identified at the different time points. (E) The expression levels of IKKε and CHEK1 proteins (left) and mRNAs (right) were examined upon IKKε knockdown in two cell lines. GAPDH was used as a loading control for immune blotting. The area of each band was quantified by ImageJ and normalized by GAPDH, and expressed as the level relative to its negative control. The mRNA levels were quantified by real-time PCR, normalized by that of GAPDH, and the expression was calculated relative to that in negative shRNA infected cells.
Figure Legend Snippet: Validation of CHEK1 lethal effects in IKKε-mtached Ovcar5 cells (A) The knockdown levels of CHEK1 by 3 different shRNAs were examined by Western blot analysis. GAPDH was used as a loading control. (B) Double-positive cells after IKKε and CHEK1 shRNA knockdown were monitored by flow cytometry after two rounds of infection. Dot plots at 7 doubling times were shown. IKKε shRNA constructs co-express LYT2 (mouse CD8α) which was detected by an antibody conjugated to APC; CHEK1 shRNA constructs co-expressed GFP. (C) Two previously validated IKKε shRNAs were used. Either IKKε #1 or IKKε #2 shRNA (in pRSM-LYT2 vector) was co-infected with one of 3 different CHEK1 shRNAs (in pRSMX-PG vector) into Ovcar5 cells. The changes in double-positive cells were measured and calculated relative to 4 doublings after infection, at which GFP signal is maximal. The average number from 3 different CHEK1 shRNA in either negative shRNA or IKKε shRNA were calculated during indicated time courses. The significant p value was calculated by a 2-sided student's t-test. (D) A2780 IKKε-matched cell lines were established and shRNA library screening performed in the same manner as in Ovcar5. Using a cut-off with a fold change of ≤ 0.7 with a p -value of ≤ 0.05 shown in bold, 3 different CHEK1 shRNAs were identified at the different time points. (E) The expression levels of IKKε and CHEK1 proteins (left) and mRNAs (right) were examined upon IKKε knockdown in two cell lines. GAPDH was used as a loading control for immune blotting. The area of each band was quantified by ImageJ and normalized by GAPDH, and expressed as the level relative to its negative control. The mRNA levels were quantified by real-time PCR, normalized by that of GAPDH, and the expression was calculated relative to that in negative shRNA infected cells.

Techniques Used: Western Blot, shRNA, Flow Cytometry, Cytometry, Infection, Construct, Plasmid Preparation, Library Screening, Expressing, Negative Control, Real-time Polymerase Chain Reaction

Pharmacological intervention of IKKε and CHEK1 in ovarian cancer cell lines (A) IKKε inhibitor (BX795) and CHEK1 inhibitor (PF477736) sensitivities were measured by XTT assay. Cells were seeded at 1000 cells/well in 50 μl and 4 replicates 16-20 hours prior to the addition of the drug. XTT assay was performed 3 days later upon drug treatment. The viability was calculated relative to no drug treated samples. (B) Cellular viability assay was performed in 2-fold serial dilutions of PF477736 (range 0 – 0.4 μM) in triplicate in a 96 well matrix format, and then cellular death (Fa; fraction affected) was calculated from XTT based viability fractions. IC50 was calculated using CompuSyn software. Shown are the results in the presence of BX795 (circle; 1.25 μM, except 0.625 μM for A2780) and the absence of the IKKε inhibitor (square). (C) IKKε and CHEK1 doubly depleted Ovcar5 and A2780 cells maintained in selective media (25 μg/ml of mycophenolic acid and 2 μg/ml of puromycin) were seeded in 96-well plates 24 hours prior to the addition of CHEK1 inhibitor PF477736. XTT assay was performed 3 days later upon drug treatment. Statistical tests were 2-sided between shNeg and shIKKε in the presence of CHEK1 inhibitor. (D) The relative cell viability was calculated based on no drug treatment, and is shown at the following suboptimal concentrations of BX795/PF477736: 1.25 μM/0.2 μM (Ovcar5, HeyA8), 1.25 μM/0.1 μM (Ovcar8, Skov3), 0.625 μM/0.1 μM (A2780), and 0.625 μM/0.05 μM (Igrov1). SD was calculated from 3 replicates. XTT assay was performed 3 days after treatment. (E) Caspase-3/7 activity was measured by Caspase-Glo assay after exposure to BX795 (2 μM) and/or PF477736 (0.5 μM) for 24 hours. Mean values ± SD of 3 replicates are shown and the data represent 4 independent experiments. (F) The cleavage of apoptotic indicator PARP-1 was examined by Western blot analysis. Cells were treated for 24 hours as the same conditions as in Figure 4E and harvested for total cell lysate preparation. Total PARP1 is shown as a loading control.
Figure Legend Snippet: Pharmacological intervention of IKKε and CHEK1 in ovarian cancer cell lines (A) IKKε inhibitor (BX795) and CHEK1 inhibitor (PF477736) sensitivities were measured by XTT assay. Cells were seeded at 1000 cells/well in 50 μl and 4 replicates 16-20 hours prior to the addition of the drug. XTT assay was performed 3 days later upon drug treatment. The viability was calculated relative to no drug treated samples. (B) Cellular viability assay was performed in 2-fold serial dilutions of PF477736 (range 0 – 0.4 μM) in triplicate in a 96 well matrix format, and then cellular death (Fa; fraction affected) was calculated from XTT based viability fractions. IC50 was calculated using CompuSyn software. Shown are the results in the presence of BX795 (circle; 1.25 μM, except 0.625 μM for A2780) and the absence of the IKKε inhibitor (square). (C) IKKε and CHEK1 doubly depleted Ovcar5 and A2780 cells maintained in selective media (25 μg/ml of mycophenolic acid and 2 μg/ml of puromycin) were seeded in 96-well plates 24 hours prior to the addition of CHEK1 inhibitor PF477736. XTT assay was performed 3 days later upon drug treatment. Statistical tests were 2-sided between shNeg and shIKKε in the presence of CHEK1 inhibitor. (D) The relative cell viability was calculated based on no drug treatment, and is shown at the following suboptimal concentrations of BX795/PF477736: 1.25 μM/0.2 μM (Ovcar5, HeyA8), 1.25 μM/0.1 μM (Ovcar8, Skov3), 0.625 μM/0.1 μM (A2780), and 0.625 μM/0.05 μM (Igrov1). SD was calculated from 3 replicates. XTT assay was performed 3 days after treatment. (E) Caspase-3/7 activity was measured by Caspase-Glo assay after exposure to BX795 (2 μM) and/or PF477736 (0.5 μM) for 24 hours. Mean values ± SD of 3 replicates are shown and the data represent 4 independent experiments. (F) The cleavage of apoptotic indicator PARP-1 was examined by Western blot analysis. Cells were treated for 24 hours as the same conditions as in Figure 4E and harvested for total cell lysate preparation. Total PARP1 is shown as a loading control.

Techniques Used: XTT Assay, Cell Viability Assay, Software, Activity Assay, Caspase-Glo Assay, Western Blot

The effect of IKKε manipulation on p21 level and its effect on survival of ovarian cancer cells (A) Ovcar5 and A2780 were depleted of IKKε or negative control and maintained under selection (25 μg/ml of mycophenolic acid). Total protein lysates were prepared to examine p21 level and the knockdown of IKKε. GAPDH was used as a loading control. (B) Caov3-wtIKKε and Caov3-IKKε K38A were maintained in selective media (100 μg/ml of G418) and total protein lysates were prepared for the protein analysis. (C) Cytoplasimc and nuclear protein fractionation was prepared using Ovcar5 and A2780 depleted for IKKε (shRNA#1) or control and maintained in culture after magnetic beads purification for LYT2 surface selection marker. (D) Ovcar5 cells were treated for 4 hours with IKKε inhibitor (BX795, 2 μM) and/or CHEK1 inhibitor (PF477736, 0.5 μM) prepared in fresh medium prior to cellular fractionation. GAPDH and Topoisomerase II were used as cytosolic and nuclear markers, respectively. (E) Cells transfected with either siNeg or si p21 were seeded at 2000 cells/well in 50 μl in 3 replicates and 8 hours later PF477736 was added in the absence or presence of BX795 (0.3 μM). XTT assay was performed 2 and 3 days later upon drug treatment. The viability was calculated relative to no drug and siNeg treated samples. The significant p values between siNeg and si p21 samples were calculated by a 2-sided student's t-test. (F) After 16 hours siRNA transfection, cells were seeded and treated for 16 hours with IKKε inhibitor (BX795, 0.3 μM) and/or CHEK1 inhibitor (PF477736, 0.4 μM) in 4 replicates. The caspase3/7 activities were shown relative to untreated siNeg samples from 4 replicates. The significant p values between siNeg and si p21 samples were calculated by a 2-sided student's t-test.
Figure Legend Snippet: The effect of IKKε manipulation on p21 level and its effect on survival of ovarian cancer cells (A) Ovcar5 and A2780 were depleted of IKKε or negative control and maintained under selection (25 μg/ml of mycophenolic acid). Total protein lysates were prepared to examine p21 level and the knockdown of IKKε. GAPDH was used as a loading control. (B) Caov3-wtIKKε and Caov3-IKKε K38A were maintained in selective media (100 μg/ml of G418) and total protein lysates were prepared for the protein analysis. (C) Cytoplasimc and nuclear protein fractionation was prepared using Ovcar5 and A2780 depleted for IKKε (shRNA#1) or control and maintained in culture after magnetic beads purification for LYT2 surface selection marker. (D) Ovcar5 cells were treated for 4 hours with IKKε inhibitor (BX795, 2 μM) and/or CHEK1 inhibitor (PF477736, 0.5 μM) prepared in fresh medium prior to cellular fractionation. GAPDH and Topoisomerase II were used as cytosolic and nuclear markers, respectively. (E) Cells transfected with either siNeg or si p21 were seeded at 2000 cells/well in 50 μl in 3 replicates and 8 hours later PF477736 was added in the absence or presence of BX795 (0.3 μM). XTT assay was performed 2 and 3 days later upon drug treatment. The viability was calculated relative to no drug and siNeg treated samples. The significant p values between siNeg and si p21 samples were calculated by a 2-sided student's t-test. (F) After 16 hours siRNA transfection, cells were seeded and treated for 16 hours with IKKε inhibitor (BX795, 0.3 μM) and/or CHEK1 inhibitor (PF477736, 0.4 μM) in 4 replicates. The caspase3/7 activities were shown relative to untreated siNeg samples from 4 replicates. The significant p values between siNeg and si p21 samples were calculated by a 2-sided student's t-test.

Techniques Used: Negative Control, Selection, Fractionation, shRNA, Magnetic Beads, Purification, Marker, Cell Fractionation, Transfection, XTT Assay

23) Product Images from "The Detailed 3D Multi-Loop Aggregate/Rosette Chromatin Architecture and Functional Dynamic Organization of the Human and Mouse Genomes"

Article Title: The Detailed 3D Multi-Loop Aggregate/Rosette Chromatin Architecture and Functional Dynamic Organization of the Human and Mouse Genomes

Journal: bioRxiv

doi: 10.1101/064642

T2C description, interaction mapping, and direct determination of the chromatin quasi-fibre and the aggregated loop/rosette 3D architecture of the human and mouse genomes: A , Cell nuclei in a population of cells (transmission light and fluorescence microscopy, Fejes-Toth et al., 2004 ) have an underlying chromatin architecture (simulated cell nucleus containing 1.2 million polymer segments; resolution 5.2 kbp, i.e. ∼50 nucleosomes; Multi-Loop-Subcompartment (MLS) rosette model with 126 kbp loops and linkers; Knoch, 2002 ). After crosslinking the DNA is restricted within the nucleus by a 1st restriction enzyme, before the cross-linked fragments are extracted and diluted such that intra fragment re-ligation is favoured. After de-crosslinking, the re-ligated material is shortened by a 2nd restriction enzyme or sonication and purified by a capture array with oligos designed next to the 1st restriction enzyme, before paired-end-sequencing over the ligation position. After alignment to the reference genome, this results in interactions frequency matrices (B-D) and scaling curves ( Fig. 2 ). B, C , Interaction matrices (logarithmic and colour coded scale; left right) of the human IGF/H19 11p 15.5-15.4 region (B) in HB2, HEK293T TEV (intact cohesin) and HEK293T HRV (cleaved cohesin) as well as the mouse β-g 7qE3-F1 region (C) for fetal brain (inactive β-globin) and liver cells (active β-globin) show the formation of subchromosomal domains separated by a linker (borders: pink lines, right; D1s, D1e: start and end of domains), which consist of loops (red lines; 8L: number of loops), representing due to the grid-like pattern loop aggregates/rosettes. A grid-like pattern is also visible in the interactions between the domains and correspond to the interactions of loops and loop bases of interacting domains. Near the diagonal the aggregation into a chromatin quasi-fibre as well as loop internal structures are visible (zooming in- and out the images can make this clearer). Between different cell types or functional states only some local differences are visible resulting in a consensus architecture and allowing simulation of the 3D architecture (middle; resolution
Figure Legend Snippet: T2C description, interaction mapping, and direct determination of the chromatin quasi-fibre and the aggregated loop/rosette 3D architecture of the human and mouse genomes: A , Cell nuclei in a population of cells (transmission light and fluorescence microscopy, Fejes-Toth et al., 2004 ) have an underlying chromatin architecture (simulated cell nucleus containing 1.2 million polymer segments; resolution 5.2 kbp, i.e. ∼50 nucleosomes; Multi-Loop-Subcompartment (MLS) rosette model with 126 kbp loops and linkers; Knoch, 2002 ). After crosslinking the DNA is restricted within the nucleus by a 1st restriction enzyme, before the cross-linked fragments are extracted and diluted such that intra fragment re-ligation is favoured. After de-crosslinking, the re-ligated material is shortened by a 2nd restriction enzyme or sonication and purified by a capture array with oligos designed next to the 1st restriction enzyme, before paired-end-sequencing over the ligation position. After alignment to the reference genome, this results in interactions frequency matrices (B-D) and scaling curves ( Fig. 2 ). B, C , Interaction matrices (logarithmic and colour coded scale; left right) of the human IGF/H19 11p 15.5-15.4 region (B) in HB2, HEK293T TEV (intact cohesin) and HEK293T HRV (cleaved cohesin) as well as the mouse β-g 7qE3-F1 region (C) for fetal brain (inactive β-globin) and liver cells (active β-globin) show the formation of subchromosomal domains separated by a linker (borders: pink lines, right; D1s, D1e: start and end of domains), which consist of loops (red lines; 8L: number of loops), representing due to the grid-like pattern loop aggregates/rosettes. A grid-like pattern is also visible in the interactions between the domains and correspond to the interactions of loops and loop bases of interacting domains. Near the diagonal the aggregation into a chromatin quasi-fibre as well as loop internal structures are visible (zooming in- and out the images can make this clearer). Between different cell types or functional states only some local differences are visible resulting in a consensus architecture and allowing simulation of the 3D architecture (middle; resolution

Techniques Used: Transmission Assay, Fluorescence, Microscopy, Ligation, Sonication, Purification, Sequencing, Functional Assay

Scaling analysis of experiments, simulations, and the DNA sequence showing the formation of a chromatin quasi-fibre and the loop aggregate/rosette genome architecture: A , The fine-structured multi-scaling resulting from the T2C interaction frequency as a function of the genomic separation for the human IGF/H19 11p 15.5-15.4 region and the mouse β-globin locus 7qE3-F1 (3 bp average (1-200 bp) and thereafter a grouping with a 1% resolution per order of magnitude which for clarity is smoothed by a running window average for > 10 3 bp; see also Fig. S8; the values
Figure Legend Snippet: Scaling analysis of experiments, simulations, and the DNA sequence showing the formation of a chromatin quasi-fibre and the loop aggregate/rosette genome architecture: A , The fine-structured multi-scaling resulting from the T2C interaction frequency as a function of the genomic separation for the human IGF/H19 11p 15.5-15.4 region and the mouse β-globin locus 7qE3-F1 (3 bp average (1-200 bp) and thereafter a grouping with a 1% resolution per order of magnitude which for clarity is smoothed by a running window average for > 10 3 bp; see also Fig. S8; the values

Techniques Used: Sequencing

24) Product Images from "Alterations in sperm long RNA contribute to the epigenetic inheritance of the effects of postnatal trauma"

Article Title: Alterations in sperm long RNA contribute to the epigenetic inheritance of the effects of postnatal trauma

Journal: Molecular psychiatry

doi: 10.1038/s41380-018-0271-6

Detection of long RNA affected by MSUS in zygotes. a Scatter plot reveals correlation of 47 mRNA and long non-coding RNA (lncRNA) with a p -value less than 0.01 (multiple testing uncorrected) in zygotes resulting from mating of MSUS males with naïve females and a q -value
Figure Legend Snippet: Detection of long RNA affected by MSUS in zygotes. a Scatter plot reveals correlation of 47 mRNA and long non-coding RNA (lncRNA) with a p -value less than 0.01 (multiple testing uncorrected) in zygotes resulting from mating of MSUS males with naïve females and a q -value

Techniques Used:

Generation of control and MSUS F1 males and of animals resulting from sperm RNA injections into zygotes. Between postnatal day (PND) 1 and 14, mouse pups are exposed to unpredictable maternal separation combined with unpredictable maternal stress (MSUS) while control mice are left undisturbed. When adult, sperm of F1 MSUS and control males is harvested, RNA is extracted, size selected then used for injection into naïve fertilized oocytes. Long RNA ( > 200 nt) includes mRNA, lncRNA, TE RNA among others. Small RNA (
Figure Legend Snippet: Generation of control and MSUS F1 males and of animals resulting from sperm RNA injections into zygotes. Between postnatal day (PND) 1 and 14, mouse pups are exposed to unpredictable maternal separation combined with unpredictable maternal stress (MSUS) while control mice are left undisturbed. When adult, sperm of F1 MSUS and control males is harvested, RNA is extracted, size selected then used for injection into naïve fertilized oocytes. Long RNA ( > 200 nt) includes mRNA, lncRNA, TE RNA among others. Small RNA (

Techniques Used: Mouse Assay, Injection

The long RNA fraction from sperm of males exposed to MSUS is sufficient to mimic some behavioural alterations observed in natural offspring of MSUS fathers. Injection of small or long sperm RNA of F1 MSUS males into naïve zygotes has no effect on ( a ) the latency to first enter an open arm on an elevated plus maze (small RNA of controls n = 25, small RNA of MSUS n = 20, Mann–Whitney U = 214.5, p > 0.05); long RNA of controls n = 17, long RNA of MSUS n = 22, Mann–Whitney U = 171, p > 0.05). Injection of small sperm RNA but not long sperm RNA decreases ( b ) time spent in the bright compartment in a light dark box (small RNA of controls n = 24, small RNA of MSUS n = 17, t (39) = 2.02 p = 0.05; Long RNA of controls n = 18, long RNA of MSUS n = 21, t (32,19) = −1.95, p > 0.05) and ( c ) time spent floating minutes 3–6 on a forced swim test (small RNA of controls n = 20, small RNA of MSUS n = 19, t (22.59) = −3.84, p = 0.001; long RNA of controls n = 16, long RNA of MSUS n = 22, Mann– Whitney U = 158, p > 0.05) in the resulting male offspring in adulthood. Data are median ± whiskers. Dots, boxes and triangles: values that lie outside the sum of the 75th percentile and 1.5 × the interquartile range or the 25th percentile minus 1.5 × the interquartile range (all values included in statistical analysis). * p
Figure Legend Snippet: The long RNA fraction from sperm of males exposed to MSUS is sufficient to mimic some behavioural alterations observed in natural offspring of MSUS fathers. Injection of small or long sperm RNA of F1 MSUS males into naïve zygotes has no effect on ( a ) the latency to first enter an open arm on an elevated plus maze (small RNA of controls n = 25, small RNA of MSUS n = 20, Mann–Whitney U = 214.5, p > 0.05); long RNA of controls n = 17, long RNA of MSUS n = 22, Mann–Whitney U = 171, p > 0.05). Injection of small sperm RNA but not long sperm RNA decreases ( b ) time spent in the bright compartment in a light dark box (small RNA of controls n = 24, small RNA of MSUS n = 17, t (39) = 2.02 p = 0.05; Long RNA of controls n = 18, long RNA of MSUS n = 21, t (32,19) = −1.95, p > 0.05) and ( c ) time spent floating minutes 3–6 on a forced swim test (small RNA of controls n = 20, small RNA of MSUS n = 19, t (22.59) = −3.84, p = 0.001; long RNA of controls n = 16, long RNA of MSUS n = 22, Mann– Whitney U = 158, p > 0.05) in the resulting male offspring in adulthood. Data are median ± whiskers. Dots, boxes and triangles: values that lie outside the sum of the 75th percentile and 1.5 × the interquartile range or the 25th percentile minus 1.5 × the interquartile range (all values included in statistical analysis). * p

Techniques Used: Injection, MANN-WHITNEY

The long RNA fraction from sperm of males exposed to MSUS is sufficient to mimic some metabolic alterations observed in natural offspring of MSUS fathers. Injection of sperm small RNA of F1 MSUS males into naïve zygotes ( a ) increases body weight (small RNA of controls n = 16, small RNA of MSUS n = 19, t (33) = 3, ** p
Figure Legend Snippet: The long RNA fraction from sperm of males exposed to MSUS is sufficient to mimic some metabolic alterations observed in natural offspring of MSUS fathers. Injection of sperm small RNA of F1 MSUS males into naïve zygotes ( a ) increases body weight (small RNA of controls n = 16, small RNA of MSUS n = 19, t (33) = 3, ** p

Techniques Used: Injection

MSUS affects sperm long RNA composition. a Heatmap showing that MSUS induces significant changes in a range of protein coding and long non-coding RNA (lncRNA) in sperm. Plotted are the row z -scores of log-normalised counts. b Heatmap showing single long intergenic non-coding (linc), RNA transcripts with differential accumulation in sperm of MSUS and control males (controls n = 4 with two biological replicates, MSUS n = 3 with three biological replicates, each replicate consists of sperm RNA pooled from five mice; multiple testing corrected p
Figure Legend Snippet: MSUS affects sperm long RNA composition. a Heatmap showing that MSUS induces significant changes in a range of protein coding and long non-coding RNA (lncRNA) in sperm. Plotted are the row z -scores of log-normalised counts. b Heatmap showing single long intergenic non-coding (linc), RNA transcripts with differential accumulation in sperm of MSUS and control males (controls n = 4 with two biological replicates, MSUS n = 3 with three biological replicates, each replicate consists of sperm RNA pooled from five mice; multiple testing corrected p

Techniques Used: Mouse Assay

Expression of long RNA transcripts affected by MSUS in sperm at different developmental spermatogenic stages and in Sertoli cells. a Density plot shows the presence of mRNA and long non-coding RNA (lncRNA) found to be differentially accumulated in MSUS sperm in spermatogonia, pachytene spermatocytes (pachytene), round spermatids and Sertoli cells. b Heatmap shows single differentially accumulated mRNA and long non-coding RNA in F1 control and MSUS sperm in comparison to spermatogonia (three samples), pachytene spermatocytes (three samples), round spermatids (three samples) and Sertoli cells (one sample) in control conditions (published data [ 26 , 27 ]). Sequencing was done once with two batches of different libraries ( a , b ). Plotted are the row z -scores of log-normalised counts
Figure Legend Snippet: Expression of long RNA transcripts affected by MSUS in sperm at different developmental spermatogenic stages and in Sertoli cells. a Density plot shows the presence of mRNA and long non-coding RNA (lncRNA) found to be differentially accumulated in MSUS sperm in spermatogonia, pachytene spermatocytes (pachytene), round spermatids and Sertoli cells. b Heatmap shows single differentially accumulated mRNA and long non-coding RNA in F1 control and MSUS sperm in comparison to spermatogonia (three samples), pachytene spermatocytes (three samples), round spermatids (three samples) and Sertoli cells (one sample) in control conditions (published data [ 26 , 27 ]). Sequencing was done once with two batches of different libraries ( a , b ). Plotted are the row z -scores of log-normalised counts

Techniques Used: Expressing, Sequencing

25) Product Images from "Ligation-anchored PCR unveils immune repertoire of TCR-beta from whole blood"

Article Title: Ligation-anchored PCR unveils immune repertoire of TCR-beta from whole blood

Journal: BMC Biotechnology

doi: 10.1186/s12896-015-0153-9

PCR method utilizing single target-specific primer to explore actively transcribed immune genes. A) , The scheme of an integrative approach to explore immune repertoire from whole blood (P-U represents a universal primer sitting on the linker, whereas P-VDJ represents a TRB-specific primer targeting the consensus sequence in the constant region right after J-segment); B) , DNA gel image of PCR amplified variable region (V-region) of TRB gene from a healthy donor under different conditions; C) , DNA gel image of amplicons from malignant and benign patients under linker ligation or control condition.
Figure Legend Snippet: PCR method utilizing single target-specific primer to explore actively transcribed immune genes. A) , The scheme of an integrative approach to explore immune repertoire from whole blood (P-U represents a universal primer sitting on the linker, whereas P-VDJ represents a TRB-specific primer targeting the consensus sequence in the constant region right after J-segment); B) , DNA gel image of PCR amplified variable region (V-region) of TRB gene from a healthy donor under different conditions; C) , DNA gel image of amplicons from malignant and benign patients under linker ligation or control condition.

Techniques Used: Polymerase Chain Reaction, Sequencing, Amplification, Ligation

26) Product Images from "Bifidobacterial Distribution Across Italian Cheeses Produced from Raw Milk"

Article Title: Bifidobacterial Distribution Across Italian Cheeses Produced from Raw Milk

Journal: Microorganisms

doi: 10.3390/microorganisms7120599

Taxonomic dissection of the raw cheese microbiota. ( a ) shows a bar plot representation of the taxonomic composition at the genus level of the profiled microbiota from cheese samples included in this study. Only taxa with relative abundance of > 1% are shown. ( b ) reports the relative abundance of bifidobacteria observed by 16S rRNA gene microbial profiling data in the 21 raw cheese samples. ( c ) depicts the bifidobacterial biodiversity, reported as the number of operational taxonomic units (OTUs), obtained from bifidobacterial internally transcribed spacer (ITS) profiling data.
Figure Legend Snippet: Taxonomic dissection of the raw cheese microbiota. ( a ) shows a bar plot representation of the taxonomic composition at the genus level of the profiled microbiota from cheese samples included in this study. Only taxa with relative abundance of > 1% are shown. ( b ) reports the relative abundance of bifidobacteria observed by 16S rRNA gene microbial profiling data in the 21 raw cheese samples. ( c ) depicts the bifidobacterial biodiversity, reported as the number of operational taxonomic units (OTUs), obtained from bifidobacterial internally transcribed spacer (ITS) profiling data.

Techniques Used: Dissection

27) Product Images from "Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation"

Article Title: Small RNA Library Preparation Method for Next-Generation Sequencing Using Chemical Modifications to Prevent Adapter Dimer Formation

Journal: PLoS ONE

doi: 10.1371/journal.pone.0167009

Comparison of NGS data between gel purified and bead purified samples within a CleanTag workflow. Libraries prepared with CleanTag small RNA library prep kit and human brain total RNA input. PCR samples were purified by gel extraction or 2-step AMPure XP bead-based protocol. Data analysis was performed with Geneious.
Figure Legend Snippet: Comparison of NGS data between gel purified and bead purified samples within a CleanTag workflow. Libraries prepared with CleanTag small RNA library prep kit and human brain total RNA input. PCR samples were purified by gel extraction or 2-step AMPure XP bead-based protocol. Data analysis was performed with Geneious.

Techniques Used: Next-Generation Sequencing, Purification, Polymerase Chain Reaction, Gel Extraction

NGS data comparison between CleanTag and TruSeq Small RNA Library Preparation Kit. Libraries prepared with TruSeq Small RNA Library Preparation Kit or CleanTag workflow with total human brain RNA input and gel purification. Samples sequenced on a HiSeq 2500 SR, 1x 100bp. Human total brain RNA at A) 100 ng, or B) 10 ng input. Data analysis performed using Geneious. Statistical analysis performed with GraphPad-One way ANOVA Turkeys multiple comparison test.
Figure Legend Snippet: NGS data comparison between CleanTag and TruSeq Small RNA Library Preparation Kit. Libraries prepared with TruSeq Small RNA Library Preparation Kit or CleanTag workflow with total human brain RNA input and gel purification. Samples sequenced on a HiSeq 2500 SR, 1x 100bp. Human total brain RNA at A) 100 ng, or B) 10 ng input. Data analysis performed using Geneious. Statistical analysis performed with GraphPad-One way ANOVA Turkeys multiple comparison test.

Techniques Used: Next-Generation Sequencing, Gel Purification

Comparison of crude and bead purified libraries using CleanTag or TruSeq small RNA library prep kit. Bioanalyzer traces of libraries prepared using 1000 ng human total brain RNA input. Crude or AMPure XP purified PCR products with A) TruSeq small RNA library preparation kit, or B) CleanTag small RNA library preparation kit.
Figure Legend Snippet: Comparison of crude and bead purified libraries using CleanTag or TruSeq small RNA library prep kit. Bioanalyzer traces of libraries prepared using 1000 ng human total brain RNA input. Crude or AMPure XP purified PCR products with A) TruSeq small RNA library preparation kit, or B) CleanTag small RNA library preparation kit.

Techniques Used: Purification, Polymerase Chain Reaction

28) Product Images from "The near-quantitative sampling of genomic DNA from various food-borne Eubacteria"

Article Title: The near-quantitative sampling of genomic DNA from various food-borne Eubacteria

Journal: BMC Microbiology

doi: 10.1186/s12866-014-0326-z

Semi-Log plot of C ∂ j ( QuickExtract ± Ampure bead clean-up) as a function of dilution. Each data point is a mean of 3 replicates ± s . These data demonstrate the poor performance of the QuickExtract protocol when used without subsequent DNA clean-up ( Ampure beads).
Figure Legend Snippet: Semi-Log plot of C ∂ j ( QuickExtract ± Ampure bead clean-up) as a function of dilution. Each data point is a mean of 3 replicates ± s . These data demonstrate the poor performance of the QuickExtract protocol when used without subsequent DNA clean-up ( Ampure beads).

Techniques Used:

29) Product Images from "The near-quantitative sampling of genomic DNA from various food-borne Eubacteria"

Article Title: The near-quantitative sampling of genomic DNA from various food-borne Eubacteria

Journal: BMC Microbiology

doi: 10.1186/s12866-014-0326-z

Semi-Log plot of C ∂ j ( QuickExtract ± Ampure bead clean-up) as a function of dilution. Each data point is a mean of 3 replicates ± s . These data demonstrate the poor performance of the QuickExtract protocol when used without subsequent DNA clean-up ( Ampure beads).
Figure Legend Snippet: Semi-Log plot of C ∂ j ( QuickExtract ± Ampure bead clean-up) as a function of dilution. Each data point is a mean of 3 replicates ± s . These data demonstrate the poor performance of the QuickExtract protocol when used without subsequent DNA clean-up ( Ampure beads).

Techniques Used:

30) Product Images from "Colonization of the human gut by bovine bacteria present in Parmesan cheese"

Article Title: Colonization of the human gut by bovine bacteria present in Parmesan cheese

Journal: Nature Communications

doi: 10.1038/s41467-019-09303-w

16S rRNA gene-based OTU analysis surveying possible microbial transmission events. Panel a shows a bar plot of the number of 16S rRNA gene OTUs shared between multiple matrices (prevalence of > 70%) collected from the same cheese making site. Panel b reports a heat map of the bacterial taxa for which OTUs have been found to be shared with a prevalence of > 70% by CF, LIT, MIL, and PC samples of the same cheese production site. Source data are provided as a Source Data file
Figure Legend Snippet: 16S rRNA gene-based OTU analysis surveying possible microbial transmission events. Panel a shows a bar plot of the number of 16S rRNA gene OTUs shared between multiple matrices (prevalence of > 70%) collected from the same cheese making site. Panel b reports a heat map of the bacterial taxa for which OTUs have been found to be shared with a prevalence of > 70% by CF, LIT, MIL, and PC samples of the same cheese production site. Source data are provided as a Source Data file

Techniques Used: Transmission Assay

16S rRNA gene-based microbial profiling of the 165 samples included in this study. Panel a visualizes the PCoA representation of the beta-diversity observed with samples colored based on matrix type: cow feces (CF), litters (LIT), milk (MIL), Parmesan cheese (PC). Panels b and c show whisker plots based on Chao1 and Shannon alpha-diversity, respectively, observed for a sub-sampling of 26,666 reads. The boxes represent 50 % of the data set, distributed between the first and third quartiles. The Median separates the boxes into the interquartile range, while the X represents the Mean. The lines extending vertically outside of the boxes show the outlier range. Source data are provided as a Source Data file
Figure Legend Snippet: 16S rRNA gene-based microbial profiling of the 165 samples included in this study. Panel a visualizes the PCoA representation of the beta-diversity observed with samples colored based on matrix type: cow feces (CF), litters (LIT), milk (MIL), Parmesan cheese (PC). Panels b and c show whisker plots based on Chao1 and Shannon alpha-diversity, respectively, observed for a sub-sampling of 26,666 reads. The boxes represent 50 % of the data set, distributed between the first and third quartiles. The Median separates the boxes into the interquartile range, while the X represents the Mean. The lines extending vertically outside of the boxes show the outlier range. Source data are provided as a Source Data file

Techniques Used: Whisker Assay, Sampling

31) Product Images from "Whole genome sequencing of Plasmodium falciparum from dried blood spots using selective whole genome amplification"

Article Title: Whole genome sequencing of Plasmodium falciparum from dried blood spots using selective whole genome amplification

Journal: Malaria Journal

doi: 10.1186/s12936-016-1641-7

Selective whole genome amplification (sWGA) enrichment. Simulated clinical samples comprising 96% human DNA and 4% P. falciparum DNA (3D7) were amplified using either WGA or sWGA. Amplified samples were sequenced to determine the proportion of reads mapping to human or P. falciparum reference genomes
Figure Legend Snippet: Selective whole genome amplification (sWGA) enrichment. Simulated clinical samples comprising 96% human DNA and 4% P. falciparum DNA (3D7) were amplified using either WGA or sWGA. Amplified samples were sequenced to determine the proportion of reads mapping to human or P. falciparum reference genomes

Techniques Used: Whole Genome Amplification, Amplification

Core genome coverage profile. Coverage depth of chromosome 1 by leucodepleted and unamplified (VB), whole genome amplified (WGA) and selective whole genome amplified (sWGA) DNA of P. falciparum strain 3D7. Black horizontal line shows positions corresponding to the core genome and red vertical line shows the centromere
Figure Legend Snippet: Core genome coverage profile. Coverage depth of chromosome 1 by leucodepleted and unamplified (VB), whole genome amplified (WGA) and selective whole genome amplified (sWGA) DNA of P. falciparum strain 3D7. Black horizontal line shows positions corresponding to the core genome and red vertical line shows the centromere

Techniques Used: Amplification, Whole Genome Amplification

32) Product Images from "Bifidobacterial Distribution Across Italian Cheeses Produced from Raw Milk"

Article Title: Bifidobacterial Distribution Across Italian Cheeses Produced from Raw Milk

Journal: Microorganisms

doi: 10.3390/microorganisms7120599

Taxonomic dissection of the raw cheese microbiota. ( a ) shows a bar plot representation of the taxonomic composition at the genus level of the profiled microbiota from cheese samples included in this study. Only taxa with relative abundance of > 1% are shown. ( b ) reports the relative abundance of bifidobacteria observed by 16S rRNA gene microbial profiling data in the 21 raw cheese samples. ( c ) depicts the bifidobacterial biodiversity, reported as the number of operational taxonomic units (OTUs), obtained from bifidobacterial internally transcribed spacer (ITS) profiling data.
Figure Legend Snippet: Taxonomic dissection of the raw cheese microbiota. ( a ) shows a bar plot representation of the taxonomic composition at the genus level of the profiled microbiota from cheese samples included in this study. Only taxa with relative abundance of > 1% are shown. ( b ) reports the relative abundance of bifidobacteria observed by 16S rRNA gene microbial profiling data in the 21 raw cheese samples. ( c ) depicts the bifidobacterial biodiversity, reported as the number of operational taxonomic units (OTUs), obtained from bifidobacterial internally transcribed spacer (ITS) profiling data.

Techniques Used: Dissection

33) Product Images from "The cytomegalovirus protein UL138 induces apoptosis of gastric cancer cells by binding to heat shock protein 70"

Article Title: The cytomegalovirus protein UL138 induces apoptosis of gastric cancer cells by binding to heat shock protein 70

Journal: Oncotarget

doi: 10.18632/oncotarget.6800

pUL138 interacts with HSP70 protein and blocks its function ( A ) The interaction between UL138 and HSP70 were screened by a human proteome microarray on pure UL138 protein. SNR indicated the average ratio of signal to noise calculated from two duplicated spots. ( B ) HSP70 forms a complex with UL138 protein detected in either UL138 or HSP70 immunoprecipitation (IP). BGC-823 cells were transiently transfected with pcDNA3.1(+)-UL138 tagged with Flag for 48 hr. Protein lysates were assayed for protein-protein interactions by IP using the specific antibodies against the UL138, Flag, or HSP70, and interacting partners were detected by immunoblotting (IB) with antibodies specific to the another protein in the pair, as indicated beside the blot. The pair protein examined in each panel is indicated above the immunoblotting. In each panel, lysates incubated with normal IgG or control serum (as negative control to UL138 specific antibody) served as negative controls. The immunoprecipitation assays are representative of 3 independent experiments. ( C ) Analysis of HSP70 and UL138 co-localization. BGC-823 were seeded on glass coverslips and transfected with pcDNA3.1(+)-UL138 or pcDNA3.1(+) and then incubated for 48 hr. Cells were stained with mouse antibody to pUL138 and rabbit antibody to HSP70. Nuclei were counterstained with DAPI. ( D ) Inhibition of UL138 in cell viability was verified by siRNA-HSP70. Cell viabilities of GC cells at 48 hr post transfection with pcDNA3.1(+)-UL138 (or pcDNA3.1(+)) and HSP70 siRNA (or non-function siRNA (siR-control)) by CCK-8 kits. Results were presented as the mean ± SEM of three individual experiments in each group. ( E ) The protein expression level of Bcl-2 was decreased when transfected with siRNA-HSP70 in BGC cell. Total protein was prepared 48 hr after transfected with HSP70 siRNA (siR-HSP70) or control siRNA (NC). HSP70 and Bcl-2 expressions were analyzed by Western blot followed by quantitated densitometric analysis using ImageJ software. GAPDH served as a loading control.
Figure Legend Snippet: pUL138 interacts with HSP70 protein and blocks its function ( A ) The interaction between UL138 and HSP70 were screened by a human proteome microarray on pure UL138 protein. SNR indicated the average ratio of signal to noise calculated from two duplicated spots. ( B ) HSP70 forms a complex with UL138 protein detected in either UL138 or HSP70 immunoprecipitation (IP). BGC-823 cells were transiently transfected with pcDNA3.1(+)-UL138 tagged with Flag for 48 hr. Protein lysates were assayed for protein-protein interactions by IP using the specific antibodies against the UL138, Flag, or HSP70, and interacting partners were detected by immunoblotting (IB) with antibodies specific to the another protein in the pair, as indicated beside the blot. The pair protein examined in each panel is indicated above the immunoblotting. In each panel, lysates incubated with normal IgG or control serum (as negative control to UL138 specific antibody) served as negative controls. The immunoprecipitation assays are representative of 3 independent experiments. ( C ) Analysis of HSP70 and UL138 co-localization. BGC-823 were seeded on glass coverslips and transfected with pcDNA3.1(+)-UL138 or pcDNA3.1(+) and then incubated for 48 hr. Cells were stained with mouse antibody to pUL138 and rabbit antibody to HSP70. Nuclei were counterstained with DAPI. ( D ) Inhibition of UL138 in cell viability was verified by siRNA-HSP70. Cell viabilities of GC cells at 48 hr post transfection with pcDNA3.1(+)-UL138 (or pcDNA3.1(+)) and HSP70 siRNA (or non-function siRNA (siR-control)) by CCK-8 kits. Results were presented as the mean ± SEM of three individual experiments in each group. ( E ) The protein expression level of Bcl-2 was decreased when transfected with siRNA-HSP70 in BGC cell. Total protein was prepared 48 hr after transfected with HSP70 siRNA (siR-HSP70) or control siRNA (NC). HSP70 and Bcl-2 expressions were analyzed by Western blot followed by quantitated densitometric analysis using ImageJ software. GAPDH served as a loading control.

Techniques Used: Microarray, Immunoprecipitation, Transfection, Incubation, Negative Control, Staining, Inhibition, CCK-8 Assay, Expressing, Western Blot, Software

Overexpression of pUL138 inhibits cell viability and induced apoptosis in different gastric cancer cell lines ( A ) Cells transfected with pcDNA3.1(+)-UL138 plasmids (UL138) and pcDNA3.1(+) plasmids (NC) were detected by Western blot at 48 hr post transfection. ( B ) Relative cell viability of GC cells when transfected with pcDNA3.1(+)-UL138 compared with pcDNA3.1(+). Cell proliferation was measured at indicated times post transfection. ( C ) Apoptosis assay by flow cytometry with annexin V-FITC/PI double-staining. GC cells transfected with pcDNA3.1(+)-UL138 present larger population of apoptosis compared with pcDNA3.1(+) at 48 hr post transfection. The dual parameter fluorescent dot plots were sorted as viable cells in the lower left quadrant, and apoptotic cells in the right quadrant. ( D ) UL138-caused inhibition of gastric cancer cells was reversed by a broad-spectrum caspase inhibitor z-VAD-FMK (ZVAD). AGS and BGC-823 cells were transfected with pcDNA3.1(+)-UL138 or pcDNA3.1(+) and ZVAD was added at the same time. At 48 hr post infection, cell proliferation was counted by a CCK-8 test and normalized by control cells (without transfection). Data was presented as means ± SEM of three independent tests. *P
Figure Legend Snippet: Overexpression of pUL138 inhibits cell viability and induced apoptosis in different gastric cancer cell lines ( A ) Cells transfected with pcDNA3.1(+)-UL138 plasmids (UL138) and pcDNA3.1(+) plasmids (NC) were detected by Western blot at 48 hr post transfection. ( B ) Relative cell viability of GC cells when transfected with pcDNA3.1(+)-UL138 compared with pcDNA3.1(+). Cell proliferation was measured at indicated times post transfection. ( C ) Apoptosis assay by flow cytometry with annexin V-FITC/PI double-staining. GC cells transfected with pcDNA3.1(+)-UL138 present larger population of apoptosis compared with pcDNA3.1(+) at 48 hr post transfection. The dual parameter fluorescent dot plots were sorted as viable cells in the lower left quadrant, and apoptotic cells in the right quadrant. ( D ) UL138-caused inhibition of gastric cancer cells was reversed by a broad-spectrum caspase inhibitor z-VAD-FMK (ZVAD). AGS and BGC-823 cells were transfected with pcDNA3.1(+)-UL138 or pcDNA3.1(+) and ZVAD was added at the same time. At 48 hr post infection, cell proliferation was counted by a CCK-8 test and normalized by control cells (without transfection). Data was presented as means ± SEM of three independent tests. *P

Techniques Used: Over Expression, Transfection, Western Blot, Apoptosis Assay, Flow Cytometry, Cytometry, Double Staining, Inhibition, Infection, CCK-8 Assay

Inductive pUL138 expression in stably UL138-transfected BGC-823 cell validates pro-apoptotic function of pUL138 ( A ) Detection of expression of pUL138 in UL138-transfected BGC-823 cells (TRE3G-UL138) by Western blot after induced with 1 μg/ml doxycycline (dox) for 48 hr. TRE3G means BGC-823 cells containing two plasmids pTRE3G and pCMV-Tet3G (BGC-TRE, cells contained two original plasmids of Tet-on system). TRE3G-UL138 means BGC-823 cells containing two plasmids pTRE3G-UL138 and pCMV-Tet3G (BGC-UL138, cells contained recombinational plasmids inductively expressing pUL138). GAPDH served as a loading control. ( B ) Cell proliferation was measured at indicated times post treating with or without dox (1 μg/ml) by CCK-8. pTRE3G-UL138 and pTRE3G referred to UL138-transfected and vector-transfected BGC-823 cell, respectively. +/−, with or without 1 μg/ml dox in cell culture medium. Proliferation was normalized to BGC-823 cells. ( C ) Apoptosis resulted by dox-induced UL138 expression was measured by flow cytometry after Annexin V-FITC/PI double-staining. Cells were treated with or without dox (+/−) at 1 μg/ml for 48 hr.
Figure Legend Snippet: Inductive pUL138 expression in stably UL138-transfected BGC-823 cell validates pro-apoptotic function of pUL138 ( A ) Detection of expression of pUL138 in UL138-transfected BGC-823 cells (TRE3G-UL138) by Western blot after induced with 1 μg/ml doxycycline (dox) for 48 hr. TRE3G means BGC-823 cells containing two plasmids pTRE3G and pCMV-Tet3G (BGC-TRE, cells contained two original plasmids of Tet-on system). TRE3G-UL138 means BGC-823 cells containing two plasmids pTRE3G-UL138 and pCMV-Tet3G (BGC-UL138, cells contained recombinational plasmids inductively expressing pUL138). GAPDH served as a loading control. ( B ) Cell proliferation was measured at indicated times post treating with or without dox (1 μg/ml) by CCK-8. pTRE3G-UL138 and pTRE3G referred to UL138-transfected and vector-transfected BGC-823 cell, respectively. +/−, with or without 1 μg/ml dox in cell culture medium. Proliferation was normalized to BGC-823 cells. ( C ) Apoptosis resulted by dox-induced UL138 expression was measured by flow cytometry after Annexin V-FITC/PI double-staining. Cells were treated with or without dox (+/−) at 1 μg/ml for 48 hr.

Techniques Used: Expressing, Stable Transfection, Transfection, Western Blot, CCK-8 Assay, Plasmid Preparation, Cell Culture, Flow Cytometry, Cytometry, Double Staining

pUL138 is associated with apoptosis-related proteins in gastric cancer cells ( A ) Differentially expressed genes (DEG) between BGC-823 cells transfected with UL138 and control vectors analyzed by mRNA microarray. P value histograms of GO terms annotated for the 500 DEGs is as indicated. The length of each bar represents the degree of P values obtained by GO analysis. The number of DEGs annotated to each GO is indicated on the left. ( B ) Expressional changes of Bcl-2, caspase-3 and caspase-9 in gastric cancer cells expressing UL138 were determined by Western blotting at 48 hr post UL138 gene transfection. AGS and BGC-823 were transfected with pcDNA3.1(+)-UL138 or pcDNA3.1(+) (indicated as UL138+/−). After 48 hr, the expression of pUL138, Bcl-2, caspase-3 and caspase-9 were determined by Western blotting followed by quantitative densitometric analysis using Image J software. Procaspase indicated caspase precursor and Cl.caspase indicated caspase cleavage. GAPDH served as a loading control. Data are presented as mean +/− SEM of 3 independent experiments. ( C ) z-VAD-FMK treatment reversed effect of UL138 overexpression. Plasmids were transfected as described in (B), then inhibited with or without z-VAD-FMK (ZVAD +/−) and analyzed as described in (B).
Figure Legend Snippet: pUL138 is associated with apoptosis-related proteins in gastric cancer cells ( A ) Differentially expressed genes (DEG) between BGC-823 cells transfected with UL138 and control vectors analyzed by mRNA microarray. P value histograms of GO terms annotated for the 500 DEGs is as indicated. The length of each bar represents the degree of P values obtained by GO analysis. The number of DEGs annotated to each GO is indicated on the left. ( B ) Expressional changes of Bcl-2, caspase-3 and caspase-9 in gastric cancer cells expressing UL138 were determined by Western blotting at 48 hr post UL138 gene transfection. AGS and BGC-823 were transfected with pcDNA3.1(+)-UL138 or pcDNA3.1(+) (indicated as UL138+/−). After 48 hr, the expression of pUL138, Bcl-2, caspase-3 and caspase-9 were determined by Western blotting followed by quantitative densitometric analysis using Image J software. Procaspase indicated caspase precursor and Cl.caspase indicated caspase cleavage. GAPDH served as a loading control. Data are presented as mean +/− SEM of 3 independent experiments. ( C ) z-VAD-FMK treatment reversed effect of UL138 overexpression. Plasmids were transfected as described in (B), then inhibited with or without z-VAD-FMK (ZVAD +/−) and analyzed as described in (B).

Techniques Used: Transfection, Microarray, Expressing, Western Blot, Software, Over Expression

Expression of pUL138 in gastric cancer cells inhibits tumor growth in vivo ( A ) The expression of UL138, caspase-3 and Bcl-2 in tumor tissue was examined by Western blotting. TRE3G-UL138 and TRE3G referred to the xenograft tumors from the stably UL138-transfected and vector-transfected BGC-823 cells, respectively. +/−, with or without dox induce. GAPDH served as a loading control. * P
Figure Legend Snippet: Expression of pUL138 in gastric cancer cells inhibits tumor growth in vivo ( A ) The expression of UL138, caspase-3 and Bcl-2 in tumor tissue was examined by Western blotting. TRE3G-UL138 and TRE3G referred to the xenograft tumors from the stably UL138-transfected and vector-transfected BGC-823 cells, respectively. +/−, with or without dox induce. GAPDH served as a loading control. * P

Techniques Used: Expressing, In Vivo, Western Blot, Stable Transfection, Transfection, Plasmid Preparation

Expression of UL138 is down-regulated in human gastric cancer tissues ( A ) UL138 transcripts were measured by quantitative real-time PCR in the tumor and paired adjacent non-neoplastic (Normal) specimens of 49 HCMV positive gastric cancer samples. The relative expression of UL138 was normalized to GAPDH (2 −ΔΔCt ). P value was obtained by using a paired Student's t -test. * P
Figure Legend Snippet: Expression of UL138 is down-regulated in human gastric cancer tissues ( A ) UL138 transcripts were measured by quantitative real-time PCR in the tumor and paired adjacent non-neoplastic (Normal) specimens of 49 HCMV positive gastric cancer samples. The relative expression of UL138 was normalized to GAPDH (2 −ΔΔCt ). P value was obtained by using a paired Student's t -test. * P

Techniques Used: Expressing, Real-time Polymerase Chain Reaction

34) Product Images from "Preclinical Characterization of GSK2336805, a Novel Inhibitor of Hepatitis C Virus Replication That Selects for Resistance in NS5A"

Article Title: Preclinical Characterization of GSK2336805, a Novel Inhibitor of Hepatitis C Virus Replication That Selects for Resistance in NS5A

Journal: Antimicrobial Agents and Chemotherapy

doi: 10.1128/AAC.01363-13

Activity of GSK2336805 on chimeric replicons containing genotype 1a and 1b NS5A patient sequences. GSK2336805 was tested in transient-transfection assays for activity on chimeric replicons containing genotype 1a or 1b patient sequences. The wild-type replicon is the genotype 1b Con-1 ET replicon, the backbone for the chimeric replicons, represented by the open circles. EC 50 s for each chimeric replicon are represented by black circles. The solid lines represent the mean EC 50 for the chimeric replicons, and the dashed lines are the 95% confidence intervals for GSK2336805 activity. Each replicon was tested at least 3 times.
Figure Legend Snippet: Activity of GSK2336805 on chimeric replicons containing genotype 1a and 1b NS5A patient sequences. GSK2336805 was tested in transient-transfection assays for activity on chimeric replicons containing genotype 1a or 1b patient sequences. The wild-type replicon is the genotype 1b Con-1 ET replicon, the backbone for the chimeric replicons, represented by the open circles. EC 50 s for each chimeric replicon are represented by black circles. The solid lines represent the mean EC 50 for the chimeric replicons, and the dashed lines are the 95% confidence intervals for GSK2336805 activity. Each replicon was tested at least 3 times.

Techniques Used: Activity Assay, Transfection

35) Product Images from "Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer"

Article Title: Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer

Journal: bioRxiv

doi: 10.1101/117143

Microbial structure of the mock communities. A and B - microbial species and respective relative proportions determined to be present in the HM782D and D6305 mock communities, respectively, following the analysis of raw data obtained from rrn amplicon sequencing in the MinION™ and chemistry R9. C and D - Comparative analysis of the expected microbial species and proportions against the data obtained after mapping of reads generated by a 4.5kbp amplicon PCR and sequenced in MinION™ device with R9 and R9.4 chemistries, for HM782D and D6305 respectively. E - Linear correlation analysis of relative read proportions obtained for all bacterial species present in HM872D and D6305 mock communities with R9 and R9.4 chemistries.
Figure Legend Snippet: Microbial structure of the mock communities. A and B - microbial species and respective relative proportions determined to be present in the HM782D and D6305 mock communities, respectively, following the analysis of raw data obtained from rrn amplicon sequencing in the MinION™ and chemistry R9. C and D - Comparative analysis of the expected microbial species and proportions against the data obtained after mapping of reads generated by a 4.5kbp amplicon PCR and sequenced in MinION™ device with R9 and R9.4 chemistries, for HM782D and D6305 respectively. E - Linear correlation analysis of relative read proportions obtained for all bacterial species present in HM872D and D6305 mock communities with R9 and R9.4 chemistries.

Techniques Used: Amplification, Sequencing, Generated, Polymerase Chain Reaction

36) Product Images from "Nucleic acid purification from plants, animals and microbes in under 30 seconds"

Article Title: Nucleic acid purification from plants, animals and microbes in under 30 seconds

Journal: PLoS Biology

doi: 10.1371/journal.pbio.2003916

Cellulose dipsticks outperform a commercially available nucleic acid purification system. (A) The time required, number of pipetting steps involved, and the costs of all consumables—including tubes and pipette tips—were calculated for purification of nucleic acids from Arabidopsis leaf tissue using either the cellulose dipstick or Agencourt AMPure paramagnetic beads. All solutions that could be prepared in advance, including lysis and wash buffers, were made and prealiquoted. The time and pipetting involved in the preparation of these solutions was not added to the tallies in the table. (B) Purified Arabidopsis DNA at different concentrations was a captured, washed, and eluted using either the cellulose dipsticks or AMPure paramagnetic beads (Beckman Coulter). The eluted DNA was used in a PCR reaction with using primers designed for the G-protein gamma subunit 1 gene. The band intensities relative to the 1 ng/μl paramagnetic bead sample appear below each band. (C) Different volumes of an Arabidopsis leaf extract were captured, washed, and eluted using either the cellulose dipsticks or AMPure paramagnetic beads and subsequently amplified in a PCR reaction as described above. The band intensities relative to the 50 μl paramagnetic bead sample appear below each band. n.a., no amplification; USD, United States Dollar.
Figure Legend Snippet: Cellulose dipsticks outperform a commercially available nucleic acid purification system. (A) The time required, number of pipetting steps involved, and the costs of all consumables—including tubes and pipette tips—were calculated for purification of nucleic acids from Arabidopsis leaf tissue using either the cellulose dipstick or Agencourt AMPure paramagnetic beads. All solutions that could be prepared in advance, including lysis and wash buffers, were made and prealiquoted. The time and pipetting involved in the preparation of these solutions was not added to the tallies in the table. (B) Purified Arabidopsis DNA at different concentrations was a captured, washed, and eluted using either the cellulose dipsticks or AMPure paramagnetic beads (Beckman Coulter). The eluted DNA was used in a PCR reaction with using primers designed for the G-protein gamma subunit 1 gene. The band intensities relative to the 1 ng/μl paramagnetic bead sample appear below each band. (C) Different volumes of an Arabidopsis leaf extract were captured, washed, and eluted using either the cellulose dipsticks or AMPure paramagnetic beads and subsequently amplified in a PCR reaction as described above. The band intensities relative to the 50 μl paramagnetic bead sample appear below each band. n.a., no amplification; USD, United States Dollar.

Techniques Used: Nucleic Acid Purification, Transferring, Purification, Lysis, Polymerase Chain Reaction, Amplification

37) Product Images from "Genotypic tropism testing by massively parallel sequencing: qualitative and quantitative analysis"

Article Title: Genotypic tropism testing by massively parallel sequencing: qualitative and quantitative analysis

Journal: BMC Medical Informatics and Decision Making

doi: 10.1186/1472-6947-11-30

Proportion of predicted X4-viruses at different FPR in ultra deep sequenced V3 amplicons (a: isolate 13 Trofile R5; b: isolate 5, Trofile X4) .
Figure Legend Snippet: Proportion of predicted X4-viruses at different FPR in ultra deep sequenced V3 amplicons (a: isolate 13 Trofile R5; b: isolate 5, Trofile X4) .

Techniques Used:

38) Product Images from "An Algorithm Measuring Donor Cell-Free DNA in Plasma of Cellular and Solid Organ Transplant Recipients That Does Not Require Donor or Recipient Genotyping"

Article Title: An Algorithm Measuring Donor Cell-Free DNA in Plasma of Cellular and Solid Organ Transplant Recipients That Does Not Require Donor or Recipient Genotyping

Journal: Frontiers in Cardiovascular Medicine

doi: 10.3389/fcvm.2016.00033

In vitro assay validation . Results for semiconductor sequencing of six separate genomic DNA mixtures (0, 0.5, 1, 2, 4, and 8%) with strong correlation ( R 2 = 0.97) between the expected (actual) and observed (measured) levels of donor cfDNA as calculated by our algorithm. The experiment was performed in triplicate with each individual admixture result plotted.
Figure Legend Snippet: In vitro assay validation . Results for semiconductor sequencing of six separate genomic DNA mixtures (0, 0.5, 1, 2, 4, and 8%) with strong correlation ( R 2 = 0.97) between the expected (actual) and observed (measured) levels of donor cfDNA as calculated by our algorithm. The experiment was performed in triplicate with each individual admixture result plotted.

Techniques Used: In Vitro, Sequencing

Removal of contaminating nuclear DNA from plasma using size selection . (A) Example of plasma cfDNA (100–200 bp) preparation containing larger fragments ( > 500 bp) of genomic DNA. (B) Contaminating gDNA was removed by incorporating a size-selection step prior to library construction. L, ladder; 1–3, plasma cfDNA samples. Green (minimum) and purple (maximum) lines indicate the sizing range of the assay.
Figure Legend Snippet: Removal of contaminating nuclear DNA from plasma using size selection . (A) Example of plasma cfDNA (100–200 bp) preparation containing larger fragments ( > 500 bp) of genomic DNA. (B) Contaminating gDNA was removed by incorporating a size-selection step prior to library construction. L, ladder; 1–3, plasma cfDNA samples. Green (minimum) and purple (maximum) lines indicate the sizing range of the assay.

Techniques Used: Selection

Effect of increasing read depth on proportion estimates . Using genomic DNA mixtures (0, 1, 2, 4, and 8%), we see that for an individual sample, read depth > 1200–1500 is associated with reduced error estimate for the proportion of donor cfDNA present. Fewer reads are associated with decreased confidence in the estimate for the proportion of donor cfDNA. Data are based upon sequencing of individual admixtures performed in triplicate.
Figure Legend Snippet: Effect of increasing read depth on proportion estimates . Using genomic DNA mixtures (0, 1, 2, 4, and 8%), we see that for an individual sample, read depth > 1200–1500 is associated with reduced error estimate for the proportion of donor cfDNA present. Fewer reads are associated with decreased confidence in the estimate for the proportion of donor cfDNA. Data are based upon sequencing of individual admixtures performed in triplicate.

Techniques Used: Sequencing

39) Product Images from "Reverse Engineering of Vaccine Antigens Using High Throughput Sequencing-enhanced mRNA Display"

Article Title: Reverse Engineering of Vaccine Antigens Using High Throughput Sequencing-enhanced mRNA Display

Journal: EBioMedicine

doi: 10.1016/j.ebiom.2015.06.021

mRNA display selection combined with HTS. The DNA library used contains a T7 promoter (T7), a CMV Translation enhancer (TE), a 15 or 27-mer random region ((Trimer) 15or27 ) and a constant region (3′ constant) encoding the peptide QLRNSCA. “Trimer” represents the mixture of 20 trimer (codon) phosphoramidites (Glen Research), each encoding one amino acid. In vitro transcription, ligation to a puromycin linker (green), in vitro translation and reverse transcription (RT) were performed as described in the Materials and methods section. mRNA/DNA–peptide fusions were applied to protein G magnetic beads (ProG beads) complexed with monoclonal antibodies (mAb) for selection. The regenerated DNA library was converted to an Illumina sequencing library by PCR amplification using forward and reverse primers containing Illumina adapters and subjected to MiSeq sequencing.
Figure Legend Snippet: mRNA display selection combined with HTS. The DNA library used contains a T7 promoter (T7), a CMV Translation enhancer (TE), a 15 or 27-mer random region ((Trimer) 15or27 ) and a constant region (3′ constant) encoding the peptide QLRNSCA. “Trimer” represents the mixture of 20 trimer (codon) phosphoramidites (Glen Research), each encoding one amino acid. In vitro transcription, ligation to a puromycin linker (green), in vitro translation and reverse transcription (RT) were performed as described in the Materials and methods section. mRNA/DNA–peptide fusions were applied to protein G magnetic beads (ProG beads) complexed with monoclonal antibodies (mAb) for selection. The regenerated DNA library was converted to an Illumina sequencing library by PCR amplification using forward and reverse primers containing Illumina adapters and subjected to MiSeq sequencing.

Techniques Used: Selection, In Vitro, Ligation, Magnetic Beads, Sequencing, Polymerase Chain Reaction, Amplification

40) Product Images from "How to Feed the Mammalian Gut Microbiota: Bacterial and Metabolic Modulation by Dietary Fibers"

Article Title: How to Feed the Mammalian Gut Microbiota: Bacterial and Metabolic Modulation by Dietary Fibers

Journal: Frontiers in Microbiology

doi: 10.3389/fmicb.2017.01749

Metabolomics profile of dietary fiber intervention on rats. (A–C) Show the PCA score plots of rat fecal samples, respectively, for I ( R 2 (cum) = 0.57, Q y(cum) 2 = 0.33), RS ( R 2 (cum) = 0.65, Q y(cum) 2 = 0.414), and CP ( R 2 (cum) = 0.70, Q y(cum) 2 = 0.408). PCA score plots were obtained considering all metabolites obtained from NMR analysis. The discriminant metabolites between pre- and post-prebiotic intake are reported on the right. Pre-treatment (T0) and post-treatment (T2) samples were considered for the analysis. (D) Shows the correlation based on a co-variance analysis depicting the relationship between fecal microbiota members, detected through 16S rRNA profiling analysis. (E) Depicts a heat map representation illustrating the co-variance between the bacterial species abundance resulting from 16S rRNA gene-based microbial profiling and the abundance of metabolites resulting from NMR analyses of rat fecal samples. p -Values are reported inside each significant square.
Figure Legend Snippet: Metabolomics profile of dietary fiber intervention on rats. (A–C) Show the PCA score plots of rat fecal samples, respectively, for I ( R 2 (cum) = 0.57, Q y(cum) 2 = 0.33), RS ( R 2 (cum) = 0.65, Q y(cum) 2 = 0.414), and CP ( R 2 (cum) = 0.70, Q y(cum) 2 = 0.408). PCA score plots were obtained considering all metabolites obtained from NMR analysis. The discriminant metabolites between pre- and post-prebiotic intake are reported on the right. Pre-treatment (T0) and post-treatment (T2) samples were considered for the analysis. (D) Shows the correlation based on a co-variance analysis depicting the relationship between fecal microbiota members, detected through 16S rRNA profiling analysis. (E) Depicts a heat map representation illustrating the co-variance between the bacterial species abundance resulting from 16S rRNA gene-based microbial profiling and the abundance of metabolites resulting from NMR analyses of rat fecal samples. p -Values are reported inside each significant square.

Techniques Used: Nuclear Magnetic Resonance

Related Articles

Amplification:

Article Title: Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer
Article Snippet: .. The adapter-ligated amplicon was recovered again using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA), washing twice with the ABB buffer supplied in the SQK-LSK1008 sequencing kit (Oxford Nanopore Technologies, Oxford, UK), and was eluted in Agencourt AMPure XP beads by adding 25 μL of elution buffer and incubating for 10 minutes at 37°C before pelleting in a magnetic rack. .. Samples for the Illumina MiSeq approach were sent to the National Center for Genomic Analaysis (CNAG, Barcelona, Spain) for multiplex sequencing in 1 lane of MiSeq instrument with 2 × 300 paired-end configuration.

Article Title: Cervicovaginal microbiome composition drives metabolic profiles in healthy pregnancy
Article Snippet: .. The amplicon library was concentrated using the Agencourt AMPure XP system (Beckman-Coulter), quantified using the KAPA Library Quantification Kit (KAPA Biosystems) and diluted to 2nM. .. Equimolar PhiX was added at 40% final volume to the amplicon library; the 16S rRNA amplicon pool was sequenced on the Illumina NextSeq 500 Platform on a 153bp x 153bp sequencing run, and the ITS2 amplicon pool was sequenced on the Illumina MiSeq platform on a 290bp x 290bp run.

Article Title: Whole genome sequencing of Plasmodium falciparum from dried blood spots using selective whole genome amplification
Article Snippet: .. Library preparation of amplified samples and short read high throughput sequencing sWGA products (≥500 ng total DNA) were cleaned using Agencourt Ampure XP beads (Beckman Coulter) following manufacturer’s instructions. .. After incubation, the tube containing bead/DNA mixture was placed on a magnetic rack to capture the DNA-bound beads while the unbound solution was discarded.

Next-Generation Sequencing:

Article Title: Whole genome sequencing of Plasmodium falciparum from dried blood spots using selective whole genome amplification
Article Snippet: .. Library preparation of amplified samples and short read high throughput sequencing sWGA products (≥500 ng total DNA) were cleaned using Agencourt Ampure XP beads (Beckman Coulter) following manufacturer’s instructions. .. After incubation, the tube containing bead/DNA mixture was placed on a magnetic rack to capture the DNA-bound beads while the unbound solution was discarded.

Article Title: The roles of hox 13 genes in newt limb development and regeneration
Article Snippet: .. Amplicons for NGS analysis were obtained by primers containing barcoded overhang adaptor sequences according to a 16S Metagenomic Sequencing Library Preparation Kit, and purified using AMPure XP (Beckman Coulter, Pasadena, CA, USA). .. All amplicons were mixed and subjected to an Illumina MiSeq run (paired-end 300; Macrogen).

Purification:

Article Title: Long-term Evaluation of AAV-CRISPR Genome Editing for Duchenne Muscular Dystrophy
Article Snippet: .. Amplicons were purified with Ampure beads (Beckman Coulter) at 1.8x. .. A short 10-cycle PCR was used to add experimental barcodes and Illumina adapter sequences.

Article Title: Loss of compensatory pro-survival and anti-apoptotic modulator, IKKε, sensitizes ovarian cancer cells to CHEK1 loss through an increased level of p21
Article Snippet: .. At this point, 1μg of each PCR product from samples No 1-8 in A2780 days 0, 4, 7 and Ovcar5 days 0, 7, 14 was combined and further purified by Agencourt AMPure XP system (Beckman Coulter) and eluted with 40 μl of ddH2 O. Thirty microliters of 1:10 diluted samples were sent for sequencing (NCI, CCR-sequencing facility). .. The sequencing was done by Illumina GA IIx with TrueSeq 2.0, alignment was done by Illumina Casava 1.8.1, and HG19 was used as a reference genome.

Article Title: Streamlined freshwater bacterioplankton Nanopelagicales (acI) and “Ca. Fonsibacter” (LD12) thrive in functional cohorts
Article Snippet: .. Amplicons were again purified using Agencourt AMPure XP purification system (Beckman Coulter, Danvers, MA, USA) then quantified using PicoGreen (Invitrogen) before pooling at equi-molar amounts for each run. ..

Sequencing:

Article Title: Multi-locus and long amplicon sequencing approach to study microbial diversity at species level using the MinION™ portable nanopore sequencer
Article Snippet: .. The adapter-ligated amplicon was recovered again using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA), washing twice with the ABB buffer supplied in the SQK-LSK1008 sequencing kit (Oxford Nanopore Technologies, Oxford, UK), and was eluted in Agencourt AMPure XP beads by adding 25 μL of elution buffer and incubating for 10 minutes at 37°C before pelleting in a magnetic rack. .. Samples for the Illumina MiSeq approach were sent to the National Center for Genomic Analaysis (CNAG, Barcelona, Spain) for multiplex sequencing in 1 lane of MiSeq instrument with 2 × 300 paired-end configuration.

Article Title: Loss of compensatory pro-survival and anti-apoptotic modulator, IKKε, sensitizes ovarian cancer cells to CHEK1 loss through an increased level of p21
Article Snippet: .. At this point, 1μg of each PCR product from samples No 1-8 in A2780 days 0, 4, 7 and Ovcar5 days 0, 7, 14 was combined and further purified by Agencourt AMPure XP system (Beckman Coulter) and eluted with 40 μl of ddH2 O. Thirty microliters of 1:10 diluted samples were sent for sequencing (NCI, CCR-sequencing facility). .. The sequencing was done by Illumina GA IIx with TrueSeq 2.0, alignment was done by Illumina Casava 1.8.1, and HG19 was used as a reference genome.

Article Title: The roles of hox 13 genes in newt limb development and regeneration
Article Snippet: .. Amplicons for NGS analysis were obtained by primers containing barcoded overhang adaptor sequences according to a 16S Metagenomic Sequencing Library Preparation Kit, and purified using AMPure XP (Beckman Coulter, Pasadena, CA, USA). .. All amplicons were mixed and subjected to an Illumina MiSeq run (paired-end 300; Macrogen).

Selection:

Article Title: Identification of novel HNF1B mRNA splicing variants and their qualitative and semi-quantitative profile in selected healthy and tumour tissues
Article Snippet: .. Size Selection In order to reduce large amplicons containing canonical exon-exon junctions ( > 150 bp), and to purify the remaining shorter amplicons, all mPCR pools were subjected to a two-step size selection using AMPure XP reagent (Beckman Coulter). .. In the first step a 1.8x reagent concentration, and in the second step a 3x reagent concentration was used.

Polymerase Chain Reaction:

Article Title: Loss of compensatory pro-survival and anti-apoptotic modulator, IKKε, sensitizes ovarian cancer cells to CHEK1 loss through an increased level of p21
Article Snippet: .. At this point, 1μg of each PCR product from samples No 1-8 in A2780 days 0, 4, 7 and Ovcar5 days 0, 7, 14 was combined and further purified by Agencourt AMPure XP system (Beckman Coulter) and eluted with 40 μl of ddH2 O. Thirty microliters of 1:10 diluted samples were sent for sequencing (NCI, CCR-sequencing facility). .. The sequencing was done by Illumina GA IIx with TrueSeq 2.0, alignment was done by Illumina Casava 1.8.1, and HG19 was used as a reference genome.

Similar Products

  • Logo
  • About
  • News
  • Press Release
  • Team
  • Advisors
  • Partners
  • Contact
  • Bioz Stars
  • Bioz vStars
  • 94
    Beckman Coulter ampure magnetic beads
    Semi-Log plot of C ∂ j ( <t>QuickExtract</t> ± <t>Ampure</t> bead clean-up) as a function of dilution. Each data point is a mean of 3 replicates ± s . These data demonstrate the poor performance of the QuickExtract protocol when used without subsequent DNA clean-up ( Ampure beads).
    Ampure Magnetic Beads, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 94/100, based on 52 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/ampure magnetic beads/product/Beckman Coulter
    Average 94 stars, based on 52 article reviews
    Price from $9.99 to $1999.99
    ampure magnetic beads - by Bioz Stars, 2020-09
    94/100 stars
      Buy from Supplier

    91
    Beckman Coulter magnetic beads nucleic acid extraction agencout ampure xp pcr purification kit
    Cellulose dipsticks outperform a commercially available nucleic acid purification system. (A) The time required, number of pipetting steps involved, and the costs of all consumables—including tubes and pipette tips—were calculated for purification of nucleic acids from Arabidopsis leaf tissue using either the cellulose dipstick or Agencourt <t>AMPure</t> paramagnetic beads. All solutions that could be prepared in advance, including lysis and wash buffers, were made and prealiquoted. The time and pipetting involved in the preparation of these solutions was not added to the tallies in the table. (B) Purified Arabidopsis DNA at different concentrations was a captured, washed, and eluted using either the cellulose dipsticks or AMPure paramagnetic beads (Beckman Coulter). The eluted DNA was used in a <t>PCR</t> reaction with using primers designed for the G-protein gamma subunit 1 gene. The band intensities relative to the 1 ng/μl paramagnetic bead sample appear below each band. (C) Different volumes of an Arabidopsis leaf extract were captured, washed, and eluted using either the cellulose dipsticks or AMPure paramagnetic beads and subsequently amplified in a PCR reaction as described above. The band intensities relative to the 50 μl paramagnetic bead sample appear below each band. n.a., no amplification; USD, United States Dollar.
    Magnetic Beads Nucleic Acid Extraction Agencout Ampure Xp Pcr Purification Kit, supplied by Beckman Coulter, used in various techniques. Bioz Stars score: 91/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/magnetic beads nucleic acid extraction agencout ampure xp pcr purification kit/product/Beckman Coulter
    Average 91 stars, based on 1 article reviews
    Price from $9.99 to $1999.99
    magnetic beads nucleic acid extraction agencout ampure xp pcr purification kit - by Bioz Stars, 2020-09
    91/100 stars
      Buy from Supplier

    Image Search Results


    Semi-Log plot of C ∂ j ( QuickExtract ± Ampure bead clean-up) as a function of dilution. Each data point is a mean of 3 replicates ± s . These data demonstrate the poor performance of the QuickExtract protocol when used without subsequent DNA clean-up ( Ampure beads).

    Journal: BMC Microbiology

    Article Title: The near-quantitative sampling of genomic DNA from various food-borne Eubacteria

    doi: 10.1186/s12866-014-0326-z

    Figure Lengend Snippet: Semi-Log plot of C ∂ j ( QuickExtract ± Ampure bead clean-up) as a function of dilution. Each data point is a mean of 3 replicates ± s . These data demonstrate the poor performance of the QuickExtract protocol when used without subsequent DNA clean-up ( Ampure beads).

    Article Snippet: This step involved adding 72 μL of AmPure magnetic beads (Agencourt Bioscience, Beverly, MA, USA) to 40 μL of the QuickExtract solution and mixing thoroughly using a pipette.

    Techniques:

    Cellulose dipsticks outperform a commercially available nucleic acid purification system. (A) The time required, number of pipetting steps involved, and the costs of all consumables—including tubes and pipette tips—were calculated for purification of nucleic acids from Arabidopsis leaf tissue using either the cellulose dipstick or Agencourt AMPure paramagnetic beads. All solutions that could be prepared in advance, including lysis and wash buffers, were made and prealiquoted. The time and pipetting involved in the preparation of these solutions was not added to the tallies in the table. (B) Purified Arabidopsis DNA at different concentrations was a captured, washed, and eluted using either the cellulose dipsticks or AMPure paramagnetic beads (Beckman Coulter). The eluted DNA was used in a PCR reaction with using primers designed for the G-protein gamma subunit 1 gene. The band intensities relative to the 1 ng/μl paramagnetic bead sample appear below each band. (C) Different volumes of an Arabidopsis leaf extract were captured, washed, and eluted using either the cellulose dipsticks or AMPure paramagnetic beads and subsequently amplified in a PCR reaction as described above. The band intensities relative to the 50 μl paramagnetic bead sample appear below each band. n.a., no amplification; USD, United States Dollar.

    Journal: PLoS Biology

    Article Title: Nucleic acid purification from plants, animals and microbes in under 30 seconds

    doi: 10.1371/journal.pbio.2003916

    Figure Lengend Snippet: Cellulose dipsticks outperform a commercially available nucleic acid purification system. (A) The time required, number of pipetting steps involved, and the costs of all consumables—including tubes and pipette tips—were calculated for purification of nucleic acids from Arabidopsis leaf tissue using either the cellulose dipstick or Agencourt AMPure paramagnetic beads. All solutions that could be prepared in advance, including lysis and wash buffers, were made and prealiquoted. The time and pipetting involved in the preparation of these solutions was not added to the tallies in the table. (B) Purified Arabidopsis DNA at different concentrations was a captured, washed, and eluted using either the cellulose dipsticks or AMPure paramagnetic beads (Beckman Coulter). The eluted DNA was used in a PCR reaction with using primers designed for the G-protein gamma subunit 1 gene. The band intensities relative to the 1 ng/μl paramagnetic bead sample appear below each band. (C) Different volumes of an Arabidopsis leaf extract were captured, washed, and eluted using either the cellulose dipsticks or AMPure paramagnetic beads and subsequently amplified in a PCR reaction as described above. The band intensities relative to the 50 μl paramagnetic bead sample appear below each band. n.a., no amplification; USD, United States Dollar.

    Article Snippet: Magnetic beads nucleic acid extraction Agencout AMPure XP PCR Purification kit (Beckman Coulter) was used to purify DNA following the manufacturer’s recommendations.

    Techniques: Nucleic Acid Purification, Transferring, Purification, Lysis, Polymerase Chain Reaction, Amplification