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hela genomic dna gdna  (New England Biolabs)


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    Structured Review

    New England Biolabs hela genomic dna gdna
    Recommended Analysis Inclusion Criteria (A) Decision tree to determine if a position should be considered for downstream analysis. (B) Sanger sequencing for orthogonal support determines suitability for downstream modification analyses. Comparison of <t>HeLa</t> biological RNA (DRS), IVT RNA (DRS), <t>gDNA</t> (Sanger sequencing), and genome reference (GRCh38). Red bars indicate exclusion from downstream analyses, and green bars indicate inclusion.
    Hela Genomic Dna Gdna, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 86/100, based on 1 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
    https://www.bioz.com/result/hela genomic dna gdna/product/New England Biolabs
    Average 86 stars, based on 1 article reviews
    hela genomic dna gdna - by Bioz Stars, 2025-02
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    Images

    1) Product Images from "Multicellular, IVT-derived, unmodified human transcriptome for nanopore-direct RNA analysis"

    Article Title: Multicellular, IVT-derived, unmodified human transcriptome for nanopore-direct RNA analysis

    Journal: GigaByte

    doi: 10.46471/gigabyte.129

    Recommended Analysis Inclusion Criteria (A) Decision tree to determine if a position should be considered for downstream analysis. (B) Sanger sequencing for orthogonal support determines suitability for downstream modification analyses. Comparison of HeLa biological RNA (DRS), IVT RNA (DRS), gDNA (Sanger sequencing), and genome reference (GRCh38). Red bars indicate exclusion from downstream analyses, and green bars indicate inclusion.
    Figure Legend Snippet: Recommended Analysis Inclusion Criteria (A) Decision tree to determine if a position should be considered for downstream analysis. (B) Sanger sequencing for orthogonal support determines suitability for downstream modification analyses. Comparison of HeLa biological RNA (DRS), IVT RNA (DRS), gDNA (Sanger sequencing), and genome reference (GRCh38). Red bars indicate exclusion from downstream analyses, and green bars indicate inclusion.

    Techniques Used: Sequencing, Modification, Comparison



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    <t>HeLa</t> strains differ in their capacity to support Alu retrotransposition. ( A ) Top panel: Schematic of the Alu retrotransposition assay . HeLa cells were co-transfected with pTMO2F3 and p Aluneo Tet . pTMO2F3 expresses ORF2p with a carboxyl terminus 3XFLAG tag (green flag). ORF2p expression is augmented by a CMV promoter (white square), the native L1 5′ UTR (grey oval), and an SV40 polyadenylation signal sequence (black lollipop). The p Aluneo Tet retrotransposition marker ( neo Tet ; orange oval) contains a backwards copy of the neomycin phosphotransferase gene interrupted by a self-splicing group I intron (loop) that is in the same transcriptional orientation as the Alu sequence. A 44 bp poly(A) tract follows the neo Tet sequence. Aluneo Tet expression is augmented by a 7SL gene enhancer (black square) and a sequence of four consecutive thymidine residues (grey square) located downstream of the 44 bp poly(A) sequence. The neomycin phosphotransferase gene can only be expressed when the Alu transcript is spliced and subsequently is inserted into <t>genomic</t> <t>DNA</t> by L1 ORF2p (Blue circle). The resultant insertions are flanked by target site duplications (purple arrows). Bottom panel: Results of Alu retrotransposition assays . Displayed are single wells of a representative six-well tissue culture plate from Alu retrotransposition assays. The HeLa strain is denoted above each image. Below each image is the average number of G418-resistant colonies per well ± standard deviation (n = number independent transfections). ( B ) Top panel: Schematic of the L1 retrotransposition assay . HeLa cells were transfected with an engineered human L1.3 construct (pJM101/L1.3) marked with a mneoI retrotransposition indicator cassette (orange oval) that consists of a backwards copy of the neomycin phosphotransferase gene interrupted by the gamma globin intron 2 sequence (inverted ‘V’) that is in the same transcriptional orientation as the L1 sequence. A CMV promoter (white square) and an SV40 polyadenylation signal sequence (black lollipop) augment the expression of pJM101/L1.3. The neomycin phosphotransferase gene can only be expressed when the L1 transcript is spliced, the L1 proteins (ORF1p [yellow circle] and ORF2p [blue circle]) associate with their encoding L1 RNA in cis , and the L1 RNA is inserted into genomic DNA. The resultant retrotransposition insertions are flanked by target site duplications (purple arrows). Bottom panel: Results of L1 retrotransposition assays . Displayed are single wells of a representative six-well tissue culture plate from L1 retrotransposition assays. The HeLa strain is denoted above each image. Below each image is the average number of G418-resistant colonies per well ± standard deviation (n = number independent transfections).
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    New England Biolabs hela genomic dna gdna
    Recommended Analysis Inclusion Criteria (A) Decision tree to determine if a position should be considered for downstream analysis. (B) Sanger sequencing for orthogonal support determines suitability for downstream modification analyses. Comparison of <t>HeLa</t> biological RNA (DRS), IVT RNA (DRS), <t>gDNA</t> (Sanger sequencing), and genome reference (GRCh38). Red bars indicate exclusion from downstream analyses, and green bars indicate inclusion.
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    Image Search Results


    HeLa strains differ in their capacity to support Alu retrotransposition. ( A ) Top panel: Schematic of the Alu retrotransposition assay . HeLa cells were co-transfected with pTMO2F3 and p Aluneo Tet . pTMO2F3 expresses ORF2p with a carboxyl terminus 3XFLAG tag (green flag). ORF2p expression is augmented by a CMV promoter (white square), the native L1 5′ UTR (grey oval), and an SV40 polyadenylation signal sequence (black lollipop). The p Aluneo Tet retrotransposition marker ( neo Tet ; orange oval) contains a backwards copy of the neomycin phosphotransferase gene interrupted by a self-splicing group I intron (loop) that is in the same transcriptional orientation as the Alu sequence. A 44 bp poly(A) tract follows the neo Tet sequence. Aluneo Tet expression is augmented by a 7SL gene enhancer (black square) and a sequence of four consecutive thymidine residues (grey square) located downstream of the 44 bp poly(A) sequence. The neomycin phosphotransferase gene can only be expressed when the Alu transcript is spliced and subsequently is inserted into genomic DNA by L1 ORF2p (Blue circle). The resultant insertions are flanked by target site duplications (purple arrows). Bottom panel: Results of Alu retrotransposition assays . Displayed are single wells of a representative six-well tissue culture plate from Alu retrotransposition assays. The HeLa strain is denoted above each image. Below each image is the average number of G418-resistant colonies per well ± standard deviation (n = number independent transfections). ( B ) Top panel: Schematic of the L1 retrotransposition assay . HeLa cells were transfected with an engineered human L1.3 construct (pJM101/L1.3) marked with a mneoI retrotransposition indicator cassette (orange oval) that consists of a backwards copy of the neomycin phosphotransferase gene interrupted by the gamma globin intron 2 sequence (inverted ‘V’) that is in the same transcriptional orientation as the L1 sequence. A CMV promoter (white square) and an SV40 polyadenylation signal sequence (black lollipop) augment the expression of pJM101/L1.3. The neomycin phosphotransferase gene can only be expressed when the L1 transcript is spliced, the L1 proteins (ORF1p [yellow circle] and ORF2p [blue circle]) associate with their encoding L1 RNA in cis , and the L1 RNA is inserted into genomic DNA. The resultant retrotransposition insertions are flanked by target site duplications (purple arrows). Bottom panel: Results of L1 retrotransposition assays . Displayed are single wells of a representative six-well tissue culture plate from L1 retrotransposition assays. The HeLa strain is denoted above each image. Below each image is the average number of G418-resistant colonies per well ± standard deviation (n = number independent transfections).

    Journal: Nucleic Acids Research

    Article Title: Variable patterns of retrotransposition in different HeLa strains provide mechanistic insights into SINE RNA mobilization processes

    doi: 10.1093/nar/gkae448

    Figure Lengend Snippet: HeLa strains differ in their capacity to support Alu retrotransposition. ( A ) Top panel: Schematic of the Alu retrotransposition assay . HeLa cells were co-transfected with pTMO2F3 and p Aluneo Tet . pTMO2F3 expresses ORF2p with a carboxyl terminus 3XFLAG tag (green flag). ORF2p expression is augmented by a CMV promoter (white square), the native L1 5′ UTR (grey oval), and an SV40 polyadenylation signal sequence (black lollipop). The p Aluneo Tet retrotransposition marker ( neo Tet ; orange oval) contains a backwards copy of the neomycin phosphotransferase gene interrupted by a self-splicing group I intron (loop) that is in the same transcriptional orientation as the Alu sequence. A 44 bp poly(A) tract follows the neo Tet sequence. Aluneo Tet expression is augmented by a 7SL gene enhancer (black square) and a sequence of four consecutive thymidine residues (grey square) located downstream of the 44 bp poly(A) sequence. The neomycin phosphotransferase gene can only be expressed when the Alu transcript is spliced and subsequently is inserted into genomic DNA by L1 ORF2p (Blue circle). The resultant insertions are flanked by target site duplications (purple arrows). Bottom panel: Results of Alu retrotransposition assays . Displayed are single wells of a representative six-well tissue culture plate from Alu retrotransposition assays. The HeLa strain is denoted above each image. Below each image is the average number of G418-resistant colonies per well ± standard deviation (n = number independent transfections). ( B ) Top panel: Schematic of the L1 retrotransposition assay . HeLa cells were transfected with an engineered human L1.3 construct (pJM101/L1.3) marked with a mneoI retrotransposition indicator cassette (orange oval) that consists of a backwards copy of the neomycin phosphotransferase gene interrupted by the gamma globin intron 2 sequence (inverted ‘V’) that is in the same transcriptional orientation as the L1 sequence. A CMV promoter (white square) and an SV40 polyadenylation signal sequence (black lollipop) augment the expression of pJM101/L1.3. The neomycin phosphotransferase gene can only be expressed when the L1 transcript is spliced, the L1 proteins (ORF1p [yellow circle] and ORF2p [blue circle]) associate with their encoding L1 RNA in cis , and the L1 RNA is inserted into genomic DNA. The resultant retrotransposition insertions are flanked by target site duplications (purple arrows). Bottom panel: Results of L1 retrotransposition assays . Displayed are single wells of a representative six-well tissue culture plate from L1 retrotransposition assays. The HeLa strain is denoted above each image. Below each image is the average number of G418-resistant colonies per well ± standard deviation (n = number independent transfections).

    Article Snippet: Briefly, HeLa genomic DNA was sheared to ∼320 bp and subjected to 2 × 150 paired-end sequencing on an Illumina HiSeq 4000, which yielded approximately 8–15× coverage for each HeLa strain.

    Techniques: Transfection, Expressing, Sequencing, Marker, Standard Deviation, Construct

    L1 ORF2p reverse transcribes Alu RNA in HeLa cells. ( A ) LEAP assay: HeLa cells were co-transfected with pTMO2F3 and p Aluneo Tet . Anti-FLAG antibodies then were used to immunoprecipitate ORF2p-3XFLAG (blue circle) and associated Aluneo Tet RNAs. For the LEAP reaction, the ORF2p-3XFLAG eluates are combined with the RACE12T primer to synthesize Aluneo Tet cDNAs. The resultant Aluneo Tet cDNAs then were amplified using PCR with primers (red arrows) complementary to sequences at the 5′ end of the RACE12T oligo and within the 3′ end of the p Aluneo Tet cDNA, yielding a LEAP product of ∼240 bp. ( B ) LEAP results . LEAP reactions were analyzed by 2% agarose gel electrophoresis. Transfection conditions and HeLa cell lines are indicated at the top of the gel image (No RNP = LEAP reaction control; H 2 O = PCR control). DNA size markers (in bp) are shown to the left of the gel. The predicted LEAP product sizes of Alu LEAP (∼240 bp; red arrow) and L1 LEAP (∼420 bp; blue arrow) reactions are indicated on the left of the gel image. The L1 LEAP products are larger due to the presence of the pCEP4 SV40 polyadenylation signal sequence at the end of the mneoI indicator in pTMF3. ( C ) L1 ORF2p western blot results . ORF2p-3XFLAG steady state levels were determined by Western blot using anti-FLAG IP eluates (top panel) and whole cell extracts (WCL) (bottom panel) derived from transfected HeLa-JVM and HeLa-HA cells. The lanes correspond to the LEAP gel image in Figure . Anti-Flag antibodies were used to detect ORF2p-3XFLAG (green arrows). Anti-S6 antibodies (red arrows) were used to detect the S6 protein loading control. Protein size standards (in kDa) are indicated to the left of the blot images. ( D ) L1 ORF2p promotes the retrotransposition of Alu and other cellular polyadenylated RNAs by distinct mechanisms. Alu RNA (green structure) associates with ribosomes by interacting with SRP9/14 (red and purple circle) to gain access to ORF2p. The tRNA -derived SINE (i.e. B2 or SINEC_Cf ; orange cruciform) localizes to ribosomes by an unknown mechanism to gain access to ORF2p. At the ribosome, newly synthesized ORF2p (blue oval) can associate (solid purple arrows) with the poly(A) tail of L1 RNA (black wavy line) or poly(A) tracts of SINE RNAs (i.e. 7SL RNA -derived and tRNA derived SINEs) to promote their retrotransposition. L1 ORF2p can also promote the retrotransposition of SVA RNA and/or other cellular poly(A)+ RNAs (e.g. ORF1mneoI and mRNAs) that may not directly associate with the ribosome (dashed purple arrow) by an ORF1p-dependent mechanism that requires elucidation. 7SL- and tRNA -derived SINE retrotransposition appears to be inhibited in nonpermissive HeLa cell lines after ORF2p associates with Alu RNA (indicated by the red ‘X’).

    Journal: Nucleic Acids Research

    Article Title: Variable patterns of retrotransposition in different HeLa strains provide mechanistic insights into SINE RNA mobilization processes

    doi: 10.1093/nar/gkae448

    Figure Lengend Snippet: L1 ORF2p reverse transcribes Alu RNA in HeLa cells. ( A ) LEAP assay: HeLa cells were co-transfected with pTMO2F3 and p Aluneo Tet . Anti-FLAG antibodies then were used to immunoprecipitate ORF2p-3XFLAG (blue circle) and associated Aluneo Tet RNAs. For the LEAP reaction, the ORF2p-3XFLAG eluates are combined with the RACE12T primer to synthesize Aluneo Tet cDNAs. The resultant Aluneo Tet cDNAs then were amplified using PCR with primers (red arrows) complementary to sequences at the 5′ end of the RACE12T oligo and within the 3′ end of the p Aluneo Tet cDNA, yielding a LEAP product of ∼240 bp. ( B ) LEAP results . LEAP reactions were analyzed by 2% agarose gel electrophoresis. Transfection conditions and HeLa cell lines are indicated at the top of the gel image (No RNP = LEAP reaction control; H 2 O = PCR control). DNA size markers (in bp) are shown to the left of the gel. The predicted LEAP product sizes of Alu LEAP (∼240 bp; red arrow) and L1 LEAP (∼420 bp; blue arrow) reactions are indicated on the left of the gel image. The L1 LEAP products are larger due to the presence of the pCEP4 SV40 polyadenylation signal sequence at the end of the mneoI indicator in pTMF3. ( C ) L1 ORF2p western blot results . ORF2p-3XFLAG steady state levels were determined by Western blot using anti-FLAG IP eluates (top panel) and whole cell extracts (WCL) (bottom panel) derived from transfected HeLa-JVM and HeLa-HA cells. The lanes correspond to the LEAP gel image in Figure . Anti-Flag antibodies were used to detect ORF2p-3XFLAG (green arrows). Anti-S6 antibodies (red arrows) were used to detect the S6 protein loading control. Protein size standards (in kDa) are indicated to the left of the blot images. ( D ) L1 ORF2p promotes the retrotransposition of Alu and other cellular polyadenylated RNAs by distinct mechanisms. Alu RNA (green structure) associates with ribosomes by interacting with SRP9/14 (red and purple circle) to gain access to ORF2p. The tRNA -derived SINE (i.e. B2 or SINEC_Cf ; orange cruciform) localizes to ribosomes by an unknown mechanism to gain access to ORF2p. At the ribosome, newly synthesized ORF2p (blue oval) can associate (solid purple arrows) with the poly(A) tail of L1 RNA (black wavy line) or poly(A) tracts of SINE RNAs (i.e. 7SL RNA -derived and tRNA derived SINEs) to promote their retrotransposition. L1 ORF2p can also promote the retrotransposition of SVA RNA and/or other cellular poly(A)+ RNAs (e.g. ORF1mneoI and mRNAs) that may not directly associate with the ribosome (dashed purple arrow) by an ORF1p-dependent mechanism that requires elucidation. 7SL- and tRNA -derived SINE retrotransposition appears to be inhibited in nonpermissive HeLa cell lines after ORF2p associates with Alu RNA (indicated by the red ‘X’).

    Article Snippet: Briefly, HeLa genomic DNA was sheared to ∼320 bp and subjected to 2 × 150 paired-end sequencing on an Illumina HiSeq 4000, which yielded approximately 8–15× coverage for each HeLa strain.

    Techniques: Transfection, Amplification, Agarose Gel Electrophoresis, Control, Sequencing, Western Blot, Derivative Assay, Synthesized

    Recommended Analysis Inclusion Criteria (A) Decision tree to determine if a position should be considered for downstream analysis. (B) Sanger sequencing for orthogonal support determines suitability for downstream modification analyses. Comparison of HeLa biological RNA (DRS), IVT RNA (DRS), gDNA (Sanger sequencing), and genome reference (GRCh38). Red bars indicate exclusion from downstream analyses, and green bars indicate inclusion.

    Journal: GigaByte

    Article Title: Multicellular, IVT-derived, unmodified human transcriptome for nanopore-direct RNA analysis

    doi: 10.46471/gigabyte.129

    Figure Lengend Snippet: Recommended Analysis Inclusion Criteria (A) Decision tree to determine if a position should be considered for downstream analysis. (B) Sanger sequencing for orthogonal support determines suitability for downstream modification analyses. Comparison of HeLa biological RNA (DRS), IVT RNA (DRS), gDNA (Sanger sequencing), and genome reference (GRCh38). Red bars indicate exclusion from downstream analyses, and green bars indicate inclusion.

    Article Snippet: We performed Sanger sequencing on HeLa genomic DNA (gDNA) to analyze putative mismatches. gDNA extraction was performed using a Monarch Genomic DNA Purification Kit (NEB, T3010S) following the Manufacturer’s protocol for cultured cells with an input of 5e6 HeLa cells.

    Techniques: Sequencing, Modification, Comparison