small rna library prep  (New England Biolabs)


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    Name:
    NEBNext Multiplex Small RNA Library Prep Set for Illumina 1 12
    Description:
    NEBNext Multiplex Small RNA Library Prep Set for Illumina 1 12 96 rxns
    Catalog Number:
    e7300l
    Price:
    4998
    Size:
    96 rxns
    Category:
    mRNA Template Preparation for PCR
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    Structured Review

    New England Biolabs small rna library prep
    NEBNext Multiplex Small RNA Library Prep Set for Illumina 1 12
    NEBNext Multiplex Small RNA Library Prep Set for Illumina 1 12 96 rxns
    https://www.bioz.com/result/small rna library prep/product/New England Biolabs
    Average 95 stars, based on 634 article reviews
    Price from $9.99 to $1999.99
    small rna library prep - by Bioz Stars, 2020-02
    95/100 stars

    Images

    1) Product Images from "No evidence for viral small RNA production and antiviral function of Argonaute 2 in human cells"

    Article Title: No evidence for viral small RNA production and antiviral function of Argonaute 2 in human cells

    Journal: Scientific Reports

    doi: 10.1038/s41598-019-50287-w

    No evidence for an RNAi suppressive function of CVB3 3A protein. ( A ) Schematic representation of the CVB3 genome with an alignment of the 3A sequence with the HEV71 3A sequence, highlighting mutations introduced into CVB3 3A. ( B ) Viral titers of passage 0 (p0) and passage 1 (p1) CVB3 virus stocks harvested from HeLa cells. Viral titers were determined by end-point serial dilution on HeLa cells. ( C ) Top panels, size profiles of small RNAs mapping to the positive viral RNA strand (red) or negative RNA strand (blue) of CVB3. Reads were mapped allowing one mismatch and normalized to the total library size and to viral RNA levels in the sample used to prepare the sequencing libraries (relative to CVB3 WT infection in each cell line). Lower panels, distribution of 21–23 nt viral small RNAs across the CVB3 genome. 5′ positions of normalized reads are plotted. ( D ) Viral titers of supernatant harvested from CVB3 WT and CVB3 D24A/K41Q infected WT or AGO2 KO HeLa and HEK293T cells at 8, 16 and 24 hpi with p0 virus. Bars represent means and SD of 3 biological replicates.
    Figure Legend Snippet: No evidence for an RNAi suppressive function of CVB3 3A protein. ( A ) Schematic representation of the CVB3 genome with an alignment of the 3A sequence with the HEV71 3A sequence, highlighting mutations introduced into CVB3 3A. ( B ) Viral titers of passage 0 (p0) and passage 1 (p1) CVB3 virus stocks harvested from HeLa cells. Viral titers were determined by end-point serial dilution on HeLa cells. ( C ) Top panels, size profiles of small RNAs mapping to the positive viral RNA strand (red) or negative RNA strand (blue) of CVB3. Reads were mapped allowing one mismatch and normalized to the total library size and to viral RNA levels in the sample used to prepare the sequencing libraries (relative to CVB3 WT infection in each cell line). Lower panels, distribution of 21–23 nt viral small RNAs across the CVB3 genome. 5′ positions of normalized reads are plotted. ( D ) Viral titers of supernatant harvested from CVB3 WT and CVB3 D24A/K41Q infected WT or AGO2 KO HeLa and HEK293T cells at 8, 16 and 24 hpi with p0 virus. Bars represent means and SD of 3 biological replicates.

    Techniques Used: Sequencing, Serial Dilution, Infection

    2) Product Images from "PNLDC1 is essential for piRNA 3′ end trimming and transposon silencing during spermatogenesis in mice"

    Article Title: PNLDC1 is essential for piRNA 3′ end trimming and transposon silencing during spermatogenesis in mice

    Journal: Nature Communications

    doi: 10.1038/s41467-017-00854-4

    Increased piRNA sizes and reduced normal piRNAs in adult Pnldc1 Mut testes. a piRNA extension in Pnldc1 Mut mice. Total RNAs from adult WT and Pnldc1 Mut (Mut-1 and Mut-2) testes were end-labeled with [ 32 P]-ATP, and detected by 15% TBE urea gel and autoradiography. Square bracket indicates extended piRNAs. b MILI-piRNA extension in Pnldc1 Mut (Mut-1) mice. Small RNAs were isolated from immunoprecipitated MILI RNPs and were end-labeled with [ 32 P]-ATP, and detected by 15% TBE urea gel and autoradiography. Western blotting was performed with anti-MILI antibody to show immunoprecipitation efficiency. Square bracket indicates extended piRNAs. M molecular weight marker. c MIWI-piRNA extension and reduction in Pnldc1 Mut (Mut-1) mice. Small RNAs were isolated from immunoprecipitated MIWI RNPs and were end-labeled with [ 32 P]-ATP, and detected by 15% TBE urea gel and autoradiography. Western blotting was performed with anti-MIWI antibody to show immunoprecipitation efficiency. Square bracket indicates extended piRNAs. M molecular weight marker. d The length distribution of small RNAs from adult WT and Pnldc1 Mut (Mut-1 and Mut-2) testicular small RNA libraries. Data were normalized by microRNA reads (21–23 nt). e The length distribution of MILI-piRNAs from adult WT and Pnldc1 Mut (Mut-1 and Mut-2) MILI-piRNA libraries. f The length distribution of MIWI-piRNAs from adult WT and Pnldc1 Mut (Mut-1 and Mut-2) MIWI-piRNA libraries
    Figure Legend Snippet: Increased piRNA sizes and reduced normal piRNAs in adult Pnldc1 Mut testes. a piRNA extension in Pnldc1 Mut mice. Total RNAs from adult WT and Pnldc1 Mut (Mut-1 and Mut-2) testes were end-labeled with [ 32 P]-ATP, and detected by 15% TBE urea gel and autoradiography. Square bracket indicates extended piRNAs. b MILI-piRNA extension in Pnldc1 Mut (Mut-1) mice. Small RNAs were isolated from immunoprecipitated MILI RNPs and were end-labeled with [ 32 P]-ATP, and detected by 15% TBE urea gel and autoradiography. Western blotting was performed with anti-MILI antibody to show immunoprecipitation efficiency. Square bracket indicates extended piRNAs. M molecular weight marker. c MIWI-piRNA extension and reduction in Pnldc1 Mut (Mut-1) mice. Small RNAs were isolated from immunoprecipitated MIWI RNPs and were end-labeled with [ 32 P]-ATP, and detected by 15% TBE urea gel and autoradiography. Western blotting was performed with anti-MIWI antibody to show immunoprecipitation efficiency. Square bracket indicates extended piRNAs. M molecular weight marker. d The length distribution of small RNAs from adult WT and Pnldc1 Mut (Mut-1 and Mut-2) testicular small RNA libraries. Data were normalized by microRNA reads (21–23 nt). e The length distribution of MILI-piRNAs from adult WT and Pnldc1 Mut (Mut-1 and Mut-2) MILI-piRNA libraries. f The length distribution of MIWI-piRNAs from adult WT and Pnldc1 Mut (Mut-1 and Mut-2) MIWI-piRNA libraries

    Techniques Used: Mouse Assay, Labeling, Autoradiography, Isolation, Immunoprecipitation, Western Blot, Molecular Weight, Marker

    3) Product Images from "Mitochondrial membrane-based initial separation of MIWI and MILI functions during pachytene piRNA biogenesis"

    Article Title: Mitochondrial membrane-based initial separation of MIWI and MILI functions during pachytene piRNA biogenesis

    Journal: Nucleic Acids Research

    doi: 10.1093/nar/gky1281

    Increased piRNA lengths and reduced piRNA levels in adult Tdrkh cKO testes. ( A ) Severe reduction of total piRNAs in Tdrkh cKO testes. Total RNA from adult WT and Tdrkh cKO testes were end-labeled with [ 32 P]-ATP and detected by 15% TBE urea gel and autoradiography. ( B ) The length distribution of small RNAs from adult WT and Tdrkh cKO testicular small RNA libraries. Data were normalized by miRNA reads (21–23nt). ( C ) Extension of MILI-piRNAs in Tdrkh cKO testes. Small RNAs were isolated from immunoprecipitated MILI-RNPs, end-labeled with [ 32 P]-ATP and detected by 15% TBE urea gel and autoradiography. Western blotting was performed using anti-MILI antibody to show immunoprecipitation efficiency. Square bracket indicates extended piRNAs. M: molecular weight marker. ( D ) Diminished MIWI-piRNAs in Tdrkh cKO testes. Small RNAs were isolated from immunoprecipitated MIWI-RNPs, end-labeled with [ 32 P]-ATP and detected by 15% TBE urea gel and autoradiography. Western blotting was performed using anti-MIWI antibody to show immunoprecipitation efficiency. M: molecular weight marker. ( E ) The length distribution of MILI-piRNAs from adult WT and Tdrkh cKO MILI-piRNA libraries. ( F ) The length distribution of MIWI-piRNAs from adult WT and Tdrkh cKO MIWI-piRNA libraries.
    Figure Legend Snippet: Increased piRNA lengths and reduced piRNA levels in adult Tdrkh cKO testes. ( A ) Severe reduction of total piRNAs in Tdrkh cKO testes. Total RNA from adult WT and Tdrkh cKO testes were end-labeled with [ 32 P]-ATP and detected by 15% TBE urea gel and autoradiography. ( B ) The length distribution of small RNAs from adult WT and Tdrkh cKO testicular small RNA libraries. Data were normalized by miRNA reads (21–23nt). ( C ) Extension of MILI-piRNAs in Tdrkh cKO testes. Small RNAs were isolated from immunoprecipitated MILI-RNPs, end-labeled with [ 32 P]-ATP and detected by 15% TBE urea gel and autoradiography. Western blotting was performed using anti-MILI antibody to show immunoprecipitation efficiency. Square bracket indicates extended piRNAs. M: molecular weight marker. ( D ) Diminished MIWI-piRNAs in Tdrkh cKO testes. Small RNAs were isolated from immunoprecipitated MIWI-RNPs, end-labeled with [ 32 P]-ATP and detected by 15% TBE urea gel and autoradiography. Western blotting was performed using anti-MIWI antibody to show immunoprecipitation efficiency. M: molecular weight marker. ( E ) The length distribution of MILI-piRNAs from adult WT and Tdrkh cKO MILI-piRNA libraries. ( F ) The length distribution of MIWI-piRNAs from adult WT and Tdrkh cKO MIWI-piRNA libraries.

    Techniques Used: Labeling, Autoradiography, Isolation, Immunoprecipitation, Western Blot, Molecular Weight, Marker

    4) Product Images from "Conditional Hfq Association with Small Noncoding RNAs in Pseudomonas aeruginosa Revealed through Comparative UV Cross-Linking Immunoprecipitation Followed by High-Throughput Sequencing"

    Article Title: Conditional Hfq Association with Small Noncoding RNAs in Pseudomonas aeruginosa Revealed through Comparative UV Cross-Linking Immunoprecipitation Followed by High-Throughput Sequencing

    Journal: mSystems

    doi: 10.1128/mSystems.00590-19

    Overview of cross-linking immunoprecipitation with high-throughput sequencing (CLIP-seq) analysis of Pseudomonas aeruginosa Hfq under planktonic and biofilm conditions. (A) Autoradiogram and Western blotting of the CLIP-enriched Hfq-RNA complex under two physiological conditions. XL+, cross-linking; XL-, non-cross-linking; P, planktonic; B, biofilm. Biological replicates are shown in Fig. S1 in the supplemental material. (B) The distribution of Hfq peaks throughout the P. aeruginosa PAO1 genome. Peaks from planktonic and biofilm conditions are highlighted in blue and red, respectively. (C) The Venn diagram shows the number of detected peaks under planktonic and biofilm conditions. Two peaks from both conditions wherein both the start and stop positions are within 40 nt are regarded as the same peak. (D) Classification of Hfq peaks into RNA classes (5′ UTR, CDS, 3′ UTR, sRNA, tRNA, and intergenic peaks). The 5′ UTRs and 3′ UTRs were annotated from TSSs validated by differential RNA-seq ( 39 ) and terminators predicted by TransTermHP ( 40 ), as well as manual curation of sRNAs from size selection sRNA-seq conducted by previous researches ( 16 , 17 ). (E) DAVID enrichment analysis of Hfq peaks from 502 and 180 mRNAs except for intergenic regions under planktonic and biofilm conditions, respectively. The results of KEGG pathway enrichment analysis are presented. Overall results are shown in Table S2 .
    Figure Legend Snippet: Overview of cross-linking immunoprecipitation with high-throughput sequencing (CLIP-seq) analysis of Pseudomonas aeruginosa Hfq under planktonic and biofilm conditions. (A) Autoradiogram and Western blotting of the CLIP-enriched Hfq-RNA complex under two physiological conditions. XL+, cross-linking; XL-, non-cross-linking; P, planktonic; B, biofilm. Biological replicates are shown in Fig. S1 in the supplemental material. (B) The distribution of Hfq peaks throughout the P. aeruginosa PAO1 genome. Peaks from planktonic and biofilm conditions are highlighted in blue and red, respectively. (C) The Venn diagram shows the number of detected peaks under planktonic and biofilm conditions. Two peaks from both conditions wherein both the start and stop positions are within 40 nt are regarded as the same peak. (D) Classification of Hfq peaks into RNA classes (5′ UTR, CDS, 3′ UTR, sRNA, tRNA, and intergenic peaks). The 5′ UTRs and 3′ UTRs were annotated from TSSs validated by differential RNA-seq ( 39 ) and terminators predicted by TransTermHP ( 40 ), as well as manual curation of sRNAs from size selection sRNA-seq conducted by previous researches ( 16 , 17 ). (E) DAVID enrichment analysis of Hfq peaks from 502 and 180 mRNAs except for intergenic regions under planktonic and biofilm conditions, respectively. The results of KEGG pathway enrichment analysis are presented. Overall results are shown in Table S2 .

    Techniques Used: Cross-linking Immunoprecipitation, Next-Generation Sequencing, Western Blot, RNA Sequencing Assay, Selection

    Normalization and comparison of cross-linking immunoprecipitation with high-throughput sequencing of Hfq between planktonic and biofilm conditions. (A) A schematic representation of the comparison of RNA affinities to Hfq between planktonic and biofilm forms. Only overlapping peaks between two phenotypes were considered. Read coverages of cross-linked samples from planktonic conditions were divided by those from biofilms. Concomitantly, read coverages of total RNA from the planktonic condition were divided by those from biofilms. To remove the effect of RNA expression from peak information, fold changes of CLIP-normalized read coverages between planktonic and biofilm conditions were divided by those of total RNA-normalized coverage. (B to D) Mean coverages normalized according to size factors of overlapping peaks detected by CLIP-seq (B) and total RNA-seq (C) between planktonic and biofilm conditions. Dashed lines denote the reference diagonal. (D) Affinity comparison of Hfq normalized peaks on overlapping RNAs between planktonic and biofilm conditions. Dashed lines denote the reference bases. Peaks from sRNAs and others are highlighted in red and gray, respectively. Significantly enriched sRNAs in planktonic (PhrS) or biofilm (PrrF1/2) cultures are indicated with circles. The correlation coefficient ρ was calculated from planktonic versus biofilm.
    Figure Legend Snippet: Normalization and comparison of cross-linking immunoprecipitation with high-throughput sequencing of Hfq between planktonic and biofilm conditions. (A) A schematic representation of the comparison of RNA affinities to Hfq between planktonic and biofilm forms. Only overlapping peaks between two phenotypes were considered. Read coverages of cross-linked samples from planktonic conditions were divided by those from biofilms. Concomitantly, read coverages of total RNA from the planktonic condition were divided by those from biofilms. To remove the effect of RNA expression from peak information, fold changes of CLIP-normalized read coverages between planktonic and biofilm conditions were divided by those of total RNA-normalized coverage. (B to D) Mean coverages normalized according to size factors of overlapping peaks detected by CLIP-seq (B) and total RNA-seq (C) between planktonic and biofilm conditions. Dashed lines denote the reference diagonal. (D) Affinity comparison of Hfq normalized peaks on overlapping RNAs between planktonic and biofilm conditions. Dashed lines denote the reference bases. Peaks from sRNAs and others are highlighted in red and gray, respectively. Significantly enriched sRNAs in planktonic (PhrS) or biofilm (PrrF1/2) cultures are indicated with circles. The correlation coefficient ρ was calculated from planktonic versus biofilm.

    Techniques Used: Cross-linking Immunoprecipitation, Next-Generation Sequencing, RNA Expression, RNA Sequencing Assay

    Related Articles

    Amplification:

    Article Title: Metformin Treatment Suppresses Melanoma Cell Growth and Motility through Modulation of microRNA Expression
    Article Snippet: Briefly, small RNA-sequencing libraries were assembled using the NEBNext small RNA library prep kit (New England Biolabs, Cat#E7300) according to the manufacturer’s protocol. .. The 3′ and 5′ adaptors were ligated to total RNA, and reverse transcription (RT) was performed; subsequently, polymerase chain reaction (PCR) amplification was performed.

    Article Title: Holo-Seq: single-cell sequencing of holo-transcriptome
    Article Snippet: The small RNA sequencing library was constructed following the manufacturer’s protocol using a NEBNext kit (NEB #E7300). .. After PCR amplification (15 cycles), the cDNA fragment from the RNA carrier was removed by Not I digestion.

    De-Phosphorylation Assay:

    Article Title: Antisense transcription licenses nascent transcripts to mediate transcriptional gene silencing
    Article Snippet: Beads were washed five times with PNK buffer, mixed with CIP (New England Biolabs) reaction system (3 µL of CIP, 40 U of RNase In [Promega] in 80 µL of dephosphorylation buffer), and then incubated for 30 min at 37°C. .. Beads were washed twice with PNK buffer supplemented with 20 mM EGTA, twice with PNK buffer, and twice with 0.1 mg/mL BSA in RNase-free water and then resuspended in 50 µL of T4 RNA ligation buffer with 5 µM 3′ adaptor (New England Biolabs, E7300S), 10% PEG 8000, and 40 U of RNase Out.

    Construct:

    Article Title: FIERY1 promotes microRNA accumulation by suppressing rRNA-derived small interfering RNAs in Arabidopsis
    Article Snippet: .. sRNA library construction and sequencing Total RNA (20 μg) from WT (Col-0), fry1-6 , and fry1-8 was resolved on a 15% urea-PAGE gel, and the sRNA fraction (15–40 nt) was excised. sRNAs in the excised gel were recovered in 0.4 M NaCl, followed by ethanol precipitation. sRNA libraries were constructed using the NEBNext Small RNA Library Prep Set for Illumina (NEB, E7300S) according to the manufacturer’s instructions. .. The libraries were pooled and sequenced to generate 75-bp single-end reads on an Illumina NextSeq CN500 platform at Berry Genomics (Beijing, China).

    Article Title: Holo-Seq: single-cell sequencing of holo-transcriptome
    Article Snippet: .. The small RNA sequencing library was constructed following the manufacturer’s protocol using a NEBNext kit (NEB #E7300). .. After PCR amplification (15 cycles), the cDNA fragment from the RNA carrier was removed by Not I digestion.

    Incubation:

    Article Title: Antisense transcription licenses nascent transcripts to mediate transcriptional gene silencing
    Article Snippet: Beads were washed five times with PNK buffer, mixed with CIP (New England Biolabs) reaction system (3 µL of CIP, 40 U of RNase In [Promega] in 80 µL of dephosphorylation buffer), and then incubated for 30 min at 37°C. .. Beads were washed twice with PNK buffer supplemented with 20 mM EGTA, twice with PNK buffer, and twice with 0.1 mg/mL BSA in RNase-free water and then resuspended in 50 µL of T4 RNA ligation buffer with 5 µM 3′ adaptor (New England Biolabs, E7300S), 10% PEG 8000, and 40 U of RNase Out.

    Article Title: SCAF4 and SCAF8, mRNA Anti-Terminator Proteins
    Article Snippet: Cells were then fractionated into soluble (cytoplasmic and nucleoplasmic) and chromatin fractions and used for FLAG immunoprecipitations as described for ‘FLAG-SCAF4 and FLAG-SCAF8 Immuno-precipitations’ using limited RNaseI digestion (1 U/mL, Thermo Fisher Scientific, AM2294) and TURBO DNase (10 U/mLThermo Fisher Scientific, AM2238) for 2 min at 37°C, followed by 5 min incubation on ice instead of benzonase treatment for chromatin extraction of RNA-protein complexes. .. Fragmented RNA was extracted using the miRNeasy kit (QIAGEN, 217004) from ProtinaseK treated SCAF4 and SCAF8 protein-RNA complexes and 1% input samples (including on-column DNase treatment), analyzed on bioanalyzer to determine the size range and amount of co-purified RNA, end-repaired using T4 PNK (NEB, M0201S), purified using AMPureXP beads (Beckman Coulter, A63881) and used for small RNA libraries preparation using NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB, E7300S and E7580S).

    Expressing:

    Article Title: Non-invasive prediction of NAFLD severity: a comprehensive, independent validation of previously postulated serum microRNA biomarkers
    Article Snippet: sRNA cDNA libraries were prepared from total serum RNA from 8 patients (4 NAFL vs 4 NASH) from the study cohort (Table ) using NEBNext® Multiplex Small RNA Library Prep Set (#E7300 y #7580; New England BioLabs®, Inc., Ipswich, MA, USA) and miRNA were eluted using Pippin Prep System (Sage Science, Inc., Beverly, MA, USA) selecting a range size of 120–200 pb. .. Datasets and additional information are deposited in the Gene Expression Omnibus (GEO) Database ( ).

    Ligation:

    Article Title: Antisense transcription licenses nascent transcripts to mediate transcriptional gene silencing
    Article Snippet: .. Beads were washed twice with PNK buffer supplemented with 20 mM EGTA, twice with PNK buffer, and twice with 0.1 mg/mL BSA in RNase-free water and then resuspended in 50 µL of T4 RNA ligation buffer with 5 µM 3′ adaptor (New England Biolabs, E7300S), 10% PEG 8000, and 40 U of RNase Out. .. Finally, 300 U of T4 RNA ligase 2 was added, and samples were incubated for 3 h at room temperature.

    Genomic Sequencing:

    Article Title: Identification of virus-encoded microRNAs in divergent Papillomaviruses
    Article Snippet: Libraries were prepared using 180 ng of pooled RNA with the NEBNext Multiplex Small RNA Library Prep Set for Illumina (E7300, New England Biolabs) according to the manufacturer’s instructions with the addition of an additional round of indexing PCR to compensate for low input RNA. .. Libraries were quantitated with the Qubit dsDNA BR assay (ThermoFisher), QC checked with the Bioanalyzer High Sensitivity assay (Agilent Technologies), combined with other barcoded libraries, and sequenced on a single lane of a SR50 run on a HiSeq 4000 (Illumina) by the Genomic Sequencing and Analysis Facility at UT Austin.

    Infection:

    Article Title: No evidence for viral small RNA production and antiviral function of Argonaute 2 in human cells
    Article Snippet: Total RNA was isolated from CVB3 infected cells at 24 hpi and from EMCV infected cells at 8 or 10 hpi using RNA-Solv reagent (Omega Biotek R630-02). .. 5 µl of the purified small RNAs was used as input for library preparation using the NEBNext Small RNA Library Prep (NEB E7300S/E7580S).

    Sequencing:

    Article Title: Dicer promotes transcription termination at sites of replication stress to maintain genome stability
    Article Snippet: Paragraph title: Illumina Sequencing (DNA and sRNA) ... Small RNA libraries were created using the NEBNext Small RNA kit (E7300).

    Article Title: Non-invasive prediction of NAFLD severity: a comprehensive, independent validation of previously postulated serum microRNA biomarkers
    Article Snippet: Paragraph title: Serum miRNAs sequencing ... sRNA cDNA libraries were prepared from total serum RNA from 8 patients (4 NAFL vs 4 NASH) from the study cohort (Table ) using NEBNext® Multiplex Small RNA Library Prep Set (#E7300 y #7580; New England BioLabs®, Inc., Ipswich, MA, USA) and miRNA were eluted using Pippin Prep System (Sage Science, Inc., Beverly, MA, USA) selecting a range size of 120–200 pb.

    Article Title: Genomic and epigenomic immunity in common bean: the unusual features of NB-LRR gene family
    Article Snippet: Paragraph title: 2.4. Small RNA library construction, sequencing and analysis ... Small RNA-seq library was performed according to NEBNext® Multiplex Small RNA Library Prep Set for Illumina instructions (#E7300S, New England Biolabs, Inc.).

    Article Title: A comprehensive profile of circulating RNAs in human serum
    Article Snippet: Small RNA-seq was performed using NEBNext® Small RNA Library Prep Set for Illumina (Cat. No E7300, New England Biolabs Inc.). .. Sequencing libraries were indexed and 12 samples were sequenced per lane of a HiSeq 2500 (Illumina).

    Article Title: FIERY1 promotes microRNA accumulation by suppressing rRNA-derived small interfering RNAs in Arabidopsis
    Article Snippet: .. sRNA library construction and sequencing Total RNA (20 μg) from WT (Col-0), fry1-6 , and fry1-8 was resolved on a 15% urea-PAGE gel, and the sRNA fraction (15–40 nt) was excised. sRNAs in the excised gel were recovered in 0.4 M NaCl, followed by ethanol precipitation. sRNA libraries were constructed using the NEBNext Small RNA Library Prep Set for Illumina (NEB, E7300S) according to the manufacturer’s instructions. .. The libraries were pooled and sequenced to generate 75-bp single-end reads on an Illumina NextSeq CN500 platform at Berry Genomics (Beijing, China).

    Article Title: SCAF4 and SCAF8, mRNA Anti-Terminator Proteins
    Article Snippet: Fragmented RNA was extracted using the miRNeasy kit (QIAGEN, 217004) from ProtinaseK treated SCAF4 and SCAF8 protein-RNA complexes and 1% input samples (including on-column DNase treatment), analyzed on bioanalyzer to determine the size range and amount of co-purified RNA, end-repaired using T4 PNK (NEB, M0201S), purified using AMPureXP beads (Beckman Coulter, A63881) and used for small RNA libraries preparation using NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB, E7300S and E7580S). .. Final cDNA libraries were gel purified and size selected for inserts between 20-80 nt, which were confirmed by bioanalyzer prior to sequencing.

    Article Title: Circulating small non-coding RNAs associated with age, sex, smoking, body mass and physical activity
    Article Snippet: Paragraph title: RNA isolation and sequencing protocols ... Small RNAseq was performed using NEBNext® Small RNA Library Prep Set for Illumina (Cat. No E7300, New England Biolabs Inc.) with a cut size on the pippin prep (Cat. No CSD3010, Sage Science) covering RNA molecules from 17 to 47 nucleotides.

    Article Title: In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs
    Article Snippet: Small RNA-seq libraries were performed according to NEBNext® Multiplex Small RNA Library Prep Set for Illumina instructions with a different bar code for each sample (#E7300S, New England Biolabs, Inc.). .. Small RNA-seq libraries are checked for their quality on DNA1000 chip using Agilent 2100 bioanalyzer (Waldbroon, Germany) before Illumina sequencing (Illumina®, California, U.S.A.).

    Article Title: The MID-PIWI module of Piwi proteins specifies nucleotide- and strand-biases of piRNAs
    Article Snippet: .. After purification by phenol-chloroform extraction, deep sequencing libraries were prepared using NEBNext Multiplex Small RNA Library Prep Set for Illumina (Cat. No. E7300) according to the manufacturer's instructions. .. Libraries were sequenced on an Illumina HiSeq platform (EMBL Heidelberg Gene Core facility).

    Article Title: Holo-Seq: single-cell sequencing of holo-transcriptome
    Article Snippet: Paragraph title: Sequencing library construction of small RNAs from a single cell and 1 ng mESCs total RNA ... The small RNA sequencing library was constructed following the manufacturer’s protocol using a NEBNext kit (NEB #E7300).

    Multiplexing:

    Article Title: In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs
    Article Snippet: Small RNA-seq libraries were performed according to NEBNext® Multiplex Small RNA Library Prep Set for Illumina instructions with a different bar code for each sample (#E7300S, New England Biolabs, Inc.). .. The SmallRNA-seq samples have been sequenced in Single-End (SE) with a read length of 100 bases and a multiplexing rate of 10 SmallRNA-seq libraries on one lane of Hiseq2000 machine to obtained around 15 millions reads/sample.

    In Vivo:

    Article Title: Identification of virus-encoded microRNAs in divergent Papillomaviruses
    Article Snippet: Paragraph title: Detection of PV miRNA from In Vivo chaffinch leg lesions ... Libraries were prepared using 180 ng of pooled RNA with the NEBNext Multiplex Small RNA Library Prep Set for Illumina (E7300, New England Biolabs) according to the manufacturer’s instructions with the addition of an additional round of indexing PCR to compensate for low input RNA.

    RNA Sequencing Assay:

    Article Title: Genomic and epigenomic immunity in common bean: the unusual features of NB-LRR gene family
    Article Snippet: .. Small RNA-seq library was performed according to NEBNext® Multiplex Small RNA Library Prep Set for Illumina instructions (#E7300S, New England Biolabs, Inc.). .. Small RNA-seq libraries were checked for their quality on DNA1000 chip using Agilent 2100 bioanalyzer (Waldbroon, Germany) before Illumina sequencing (Illumina® California, U.S.A.).

    Article Title: A comprehensive profile of circulating RNAs in human serum
    Article Snippet: .. Small RNA-seq was performed using NEBNext® Small RNA Library Prep Set for Illumina (Cat. No E7300, New England Biolabs Inc.). .. Size selection was performed using a 3% Agarose Gel Cassette (Cat. No CSD3010) on a Pippin Prep (Sage Science) with a cut size optimized to cover RNA molecules from 17 to 47 nt in length.

    Article Title: Identification of virus-encoded microRNAs in divergent Papillomaviruses
    Article Snippet: Pooled small RNA-seq libraries were prepared from RNA harvested from either foot lesions or pectoral muscle from animals CF180/09 and CF229/12. .. Libraries were prepared using 180 ng of pooled RNA with the NEBNext Multiplex Small RNA Library Prep Set for Illumina (E7300, New England Biolabs) according to the manufacturer’s instructions with the addition of an additional round of indexing PCR to compensate for low input RNA.

    Article Title: In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs
    Article Snippet: .. Small RNA-seq libraries were performed according to NEBNext® Multiplex Small RNA Library Prep Set for Illumina instructions with a different bar code for each sample (#E7300S, New England Biolabs, Inc.). .. Small RNA-seq libraries are checked for their quality on DNA1000 chip using Agilent 2100 bioanalyzer (Waldbroon, Germany) before Illumina sequencing (Illumina®, California, U.S.A.).

    Article Title: Metformin Treatment Suppresses Melanoma Cell Growth and Motility through Modulation of microRNA Expression
    Article Snippet: .. Briefly, small RNA-sequencing libraries were assembled using the NEBNext small RNA library prep kit (New England Biolabs, Cat#E7300) according to the manufacturer’s protocol. .. The 3′ and 5′ adaptors were ligated to total RNA, and reverse transcription (RT) was performed; subsequently, polymerase chain reaction (PCR) amplification was performed.

    Sensitive Assay:

    Article Title: Identification of virus-encoded microRNAs in divergent Papillomaviruses
    Article Snippet: Libraries were prepared using 180 ng of pooled RNA with the NEBNext Multiplex Small RNA Library Prep Set for Illumina (E7300, New England Biolabs) according to the manufacturer’s instructions with the addition of an additional round of indexing PCR to compensate for low input RNA. .. Libraries were quantitated with the Qubit dsDNA BR assay (ThermoFisher), QC checked with the Bioanalyzer High Sensitivity assay (Agilent Technologies), combined with other barcoded libraries, and sequenced on a single lane of a SR50 run on a HiSeq 4000 (Illumina) by the Genomic Sequencing and Analysis Facility at UT Austin.

    Magnetic Beads:

    Article Title: Antisense transcription licenses nascent transcripts to mediate transcriptional gene silencing
    Article Snippet: Next, 35 µL of Flag resin suspension (anti-Flag M2 magnetic beads, Sigma) in RIP buffer was added to the supernatants, and samples were incubated for 12 h at 4°C. .. Beads were washed twice with PNK buffer supplemented with 20 mM EGTA, twice with PNK buffer, and twice with 0.1 mg/mL BSA in RNase-free water and then resuspended in 50 µL of T4 RNA ligation buffer with 5 µM 3′ adaptor (New England Biolabs, E7300S), 10% PEG 8000, and 40 U of RNase Out.

    Isolation:

    Article Title: Circulating small non-coding RNAs associated with age, sex, smoking, body mass and physical activity
    Article Snippet: Paragraph title: RNA isolation and sequencing protocols ... Small RNAseq was performed using NEBNext® Small RNA Library Prep Set for Illumina (Cat. No E7300, New England Biolabs Inc.) with a cut size on the pippin prep (Cat. No CSD3010, Sage Science) covering RNA molecules from 17 to 47 nucleotides.

    Article Title: In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs
    Article Snippet: RNA samples enriched for small fractions were obtained with mirVana™ miRNA Isolation Kit (#AM1560, Ambion®/Life Technologies Corporation). .. Small RNA-seq libraries were performed according to NEBNext® Multiplex Small RNA Library Prep Set for Illumina instructions with a different bar code for each sample (#E7300S, New England Biolabs, Inc.).

    Article Title: The MID-PIWI module of Piwi proteins specifies nucleotide- and strand-biases of piRNAs
    Article Snippet: Nucleic acids were isolated from immunoprecipitated HA-tagged protein complexes and resolved by 15% urea-PAGE. .. After purification by phenol-chloroform extraction, deep sequencing libraries were prepared using NEBNext Multiplex Small RNA Library Prep Set for Illumina (Cat. No. E7300) according to the manufacturer's instructions.

    Article Title: No evidence for viral small RNA production and antiviral function of Argonaute 2 in human cells
    Article Snippet: Total RNA was isolated from CVB3 infected cells at 24 hpi and from EMCV infected cells at 8 or 10 hpi using RNA-Solv reagent (Omega Biotek R630-02). .. 5 µl of the purified small RNAs was used as input for library preparation using the NEBNext Small RNA Library Prep (NEB E7300S/E7580S).

    Multiplex Assay:

    Article Title: Non-invasive prediction of NAFLD severity: a comprehensive, independent validation of previously postulated serum microRNA biomarkers
    Article Snippet: .. sRNA cDNA libraries were prepared from total serum RNA from 8 patients (4 NAFL vs 4 NASH) from the study cohort (Table ) using NEBNext® Multiplex Small RNA Library Prep Set (#E7300 y #7580; New England BioLabs®, Inc., Ipswich, MA, USA) and miRNA were eluted using Pippin Prep System (Sage Science, Inc., Beverly, MA, USA) selecting a range size of 120–200 pb. .. These libraries were sequenced in a NextSeq 500 System using a 150 cycles Kit (Illumina, Inc., San Diego, CA, USA).

    Article Title: Genomic and epigenomic immunity in common bean: the unusual features of NB-LRR gene family
    Article Snippet: .. Small RNA-seq library was performed according to NEBNext® Multiplex Small RNA Library Prep Set for Illumina instructions (#E7300S, New England Biolabs, Inc.). .. Small RNA-seq libraries were checked for their quality on DNA1000 chip using Agilent 2100 bioanalyzer (Waldbroon, Germany) before Illumina sequencing (Illumina® California, U.S.A.).

    Article Title: Identification of virus-encoded microRNAs in divergent Papillomaviruses
    Article Snippet: .. Libraries were prepared using 180 ng of pooled RNA with the NEBNext Multiplex Small RNA Library Prep Set for Illumina (E7300, New England Biolabs) according to the manufacturer’s instructions with the addition of an additional round of indexing PCR to compensate for low input RNA. .. Libraries were quantitated with the Qubit dsDNA BR assay (ThermoFisher), QC checked with the Bioanalyzer High Sensitivity assay (Agilent Technologies), combined with other barcoded libraries, and sequenced on a single lane of a SR50 run on a HiSeq 4000 (Illumina) by the Genomic Sequencing and Analysis Facility at UT Austin.

    Article Title: Chromatin Profiles of Chromosomally Integrated Human Herpesvirus-6A
    Article Snippet: .. MicroRNA (miRNA) libraries were prepared using NEBNext Multiplex Small RNA Sample Prep for Illumina (#E7300) following the manufacturer’s protocol. .. Library quantification and quality were assessed as described above, and pooled libraries were sequenced on an Illumina HiSeq 2,500 at the Genomics Core Facility (University of Texas Health Science Center, San Antonio, TX) to obtain 50-bp single-end reads. miRNA data were processed using the Oasis2 package ( ).

    Article Title: SCAF4 and SCAF8, mRNA Anti-Terminator Proteins
    Article Snippet: .. Fragmented RNA was extracted using the miRNeasy kit (QIAGEN, 217004) from ProtinaseK treated SCAF4 and SCAF8 protein-RNA complexes and 1% input samples (including on-column DNase treatment), analyzed on bioanalyzer to determine the size range and amount of co-purified RNA, end-repaired using T4 PNK (NEB, M0201S), purified using AMPureXP beads (Beckman Coulter, A63881) and used for small RNA libraries preparation using NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB, E7300S and E7580S). .. Final cDNA libraries were gel purified and size selected for inserts between 20-80 nt, which were confirmed by bioanalyzer prior to sequencing.

    Article Title: In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs
    Article Snippet: .. Small RNA-seq libraries were performed according to NEBNext® Multiplex Small RNA Library Prep Set for Illumina instructions with a different bar code for each sample (#E7300S, New England Biolabs, Inc.). .. Small RNA-seq libraries are checked for their quality on DNA1000 chip using Agilent 2100 bioanalyzer (Waldbroon, Germany) before Illumina sequencing (Illumina®, California, U.S.A.).

    Article Title: The MID-PIWI module of Piwi proteins specifies nucleotide- and strand-biases of piRNAs
    Article Snippet: .. After purification by phenol-chloroform extraction, deep sequencing libraries were prepared using NEBNext Multiplex Small RNA Library Prep Set for Illumina (Cat. No. E7300) according to the manufacturer's instructions. .. Libraries were sequenced on an Illumina HiSeq platform (EMBL Heidelberg Gene Core facility).

    Purification:

    Article Title: Chromatin Profiles of Chromosomally Integrated Human Herpesvirus-6A
    Article Snippet: MicroRNA-seq RNA was extracted and purified from iciHHV-6A, and TPA or DMSO treated 293-HHV-6A cells as described above. .. MicroRNA (miRNA) libraries were prepared using NEBNext Multiplex Small RNA Sample Prep for Illumina (#E7300) following the manufacturer’s protocol.

    Article Title: SCAF4 and SCAF8, mRNA Anti-Terminator Proteins
    Article Snippet: .. Fragmented RNA was extracted using the miRNeasy kit (QIAGEN, 217004) from ProtinaseK treated SCAF4 and SCAF8 protein-RNA complexes and 1% input samples (including on-column DNase treatment), analyzed on bioanalyzer to determine the size range and amount of co-purified RNA, end-repaired using T4 PNK (NEB, M0201S), purified using AMPureXP beads (Beckman Coulter, A63881) and used for small RNA libraries preparation using NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB, E7300S and E7580S). .. Final cDNA libraries were gel purified and size selected for inserts between 20-80 nt, which were confirmed by bioanalyzer prior to sequencing.

    Article Title: The MID-PIWI module of Piwi proteins specifies nucleotide- and strand-biases of piRNAs
    Article Snippet: .. After purification by phenol-chloroform extraction, deep sequencing libraries were prepared using NEBNext Multiplex Small RNA Library Prep Set for Illumina (Cat. No. E7300) according to the manufacturer's instructions. .. Libraries were sequenced on an Illumina HiSeq platform (EMBL Heidelberg Gene Core facility).

    Article Title: No evidence for viral small RNA production and antiviral function of Argonaute 2 in human cells
    Article Snippet: .. 5 µl of the purified small RNAs was used as input for library preparation using the NEBNext Small RNA Library Prep (NEB E7300S/E7580S). .. PCR products were size purified on a 6% acrylamide/1x TBE gel and quantified using the Agilent 2100 Bioanalyzer System.

    Article Title: Metformin Treatment Suppresses Melanoma Cell Growth and Motility through Modulation of microRNA Expression
    Article Snippet: Briefly, small RNA-sequencing libraries were assembled using the NEBNext small RNA library prep kit (New England Biolabs, Cat#E7300) according to the manufacturer’s protocol. .. PCR products were size-fractionated through 6% polyacrylamide gel electrophoresis, and bands containing 140-nucleotide RNA fragments were selected for purification.

    Article Title: Holo-Seq: single-cell sequencing of holo-transcriptome
    Article Snippet: The small RNA sequencing library was constructed following the manufacturer’s protocol using a NEBNext kit (NEB #E7300). .. After Not I digestion, the library DNA was purified for deep sequencing (more details in “Step-by-step Holo-Seq protocols” (Additional file )).

    Polymerase Chain Reaction:

    Article Title: Identification of virus-encoded microRNAs in divergent Papillomaviruses
    Article Snippet: .. Libraries were prepared using 180 ng of pooled RNA with the NEBNext Multiplex Small RNA Library Prep Set for Illumina (E7300, New England Biolabs) according to the manufacturer’s instructions with the addition of an additional round of indexing PCR to compensate for low input RNA. .. Libraries were quantitated with the Qubit dsDNA BR assay (ThermoFisher), QC checked with the Bioanalyzer High Sensitivity assay (Agilent Technologies), combined with other barcoded libraries, and sequenced on a single lane of a SR50 run on a HiSeq 4000 (Illumina) by the Genomic Sequencing and Analysis Facility at UT Austin.

    Article Title: No evidence for viral small RNA production and antiviral function of Argonaute 2 in human cells
    Article Snippet: 5 µl of the purified small RNAs was used as input for library preparation using the NEBNext Small RNA Library Prep (NEB E7300S/E7580S). .. PCR products were size purified on a 6% acrylamide/1x TBE gel and quantified using the Agilent 2100 Bioanalyzer System.

    Article Title: Metformin Treatment Suppresses Melanoma Cell Growth and Motility through Modulation of microRNA Expression
    Article Snippet: Briefly, small RNA-sequencing libraries were assembled using the NEBNext small RNA library prep kit (New England Biolabs, Cat#E7300) according to the manufacturer’s protocol. .. The 3′ and 5′ adaptors were ligated to total RNA, and reverse transcription (RT) was performed; subsequently, polymerase chain reaction (PCR) amplification was performed.

    Article Title: Holo-Seq: single-cell sequencing of holo-transcriptome
    Article Snippet: The small RNA sequencing library was constructed following the manufacturer’s protocol using a NEBNext kit (NEB #E7300). .. After PCR amplification (15 cycles), the cDNA fragment from the RNA carrier was removed by Not I digestion.

    Polyacrylamide Gel Electrophoresis:

    Article Title: Metformin Treatment Suppresses Melanoma Cell Growth and Motility through Modulation of microRNA Expression
    Article Snippet: Briefly, small RNA-sequencing libraries were assembled using the NEBNext small RNA library prep kit (New England Biolabs, Cat#E7300) according to the manufacturer’s protocol. .. PCR products were size-fractionated through 6% polyacrylamide gel electrophoresis, and bands containing 140-nucleotide RNA fragments were selected for purification.

    Agarose Gel Electrophoresis:

    Article Title: A comprehensive profile of circulating RNAs in human serum
    Article Snippet: Small RNA-seq was performed using NEBNext® Small RNA Library Prep Set for Illumina (Cat. No E7300, New England Biolabs Inc.). .. Size selection was performed using a 3% Agarose Gel Cassette (Cat. No CSD3010) on a Pippin Prep (Sage Science) with a cut size optimized to cover RNA molecules from 17 to 47 nt in length.

    Chloramphenicol Acetyltransferase Assay:

    Article Title: Metformin Treatment Suppresses Melanoma Cell Growth and Motility through Modulation of microRNA Expression
    Article Snippet: .. Briefly, small RNA-sequencing libraries were assembled using the NEBNext small RNA library prep kit (New England Biolabs, CatE7300) according to the manufacturer’s protocol. .. The 3′ and 5′ adaptors were ligated to total RNA, and reverse transcription (RT) was performed; subsequently, polymerase chain reaction (PCR) amplification was performed.

    Silver Staining:

    Article Title: SCAF4 and SCAF8, mRNA Anti-Terminator Proteins
    Article Snippet: 10% of samples were used for silver stain to verify IP and the remaining 90% treated with 4 mg/mL ProtinaseK (Sigma-Aldrich, 3115887001) with the addition of 2% SDS to the FLAG elution buffer for 30 min at 50°C. .. Fragmented RNA was extracted using the miRNeasy kit (QIAGEN, 217004) from ProtinaseK treated SCAF4 and SCAF8 protein-RNA complexes and 1% input samples (including on-column DNase treatment), analyzed on bioanalyzer to determine the size range and amount of co-purified RNA, end-repaired using T4 PNK (NEB, M0201S), purified using AMPureXP beads (Beckman Coulter, A63881) and used for small RNA libraries preparation using NEBNext Multiplex Small RNA Library Prep Set for Illumina (NEB, E7300S and E7580S).

    Chromatin Immunoprecipitation:

    Article Title: Genomic and epigenomic immunity in common bean: the unusual features of NB-LRR gene family
    Article Snippet: Small RNA library construction, sequencing and analysis RNA samples enriched for small fractions of G19833 young leaves were obtained with miRNeasy Mini Kit (#217004, QIAGEN® ) and checked for their integrity on RNANano chip, using Agilent 2100 bioanalyzer (Agilent Technologies, Waldbroon, Germany). .. Small RNA-seq library was performed according to NEBNext® Multiplex Small RNA Library Prep Set for Illumina instructions (#E7300S, New England Biolabs, Inc.).

    Article Title: In plants, decapping prevents RDR6-dependent production of small interfering RNAs from endogenous mRNAs
    Article Snippet: They were checked for their integrity on RNANano chip, using Agilent 2100 bioanalyzer (Agilent Technologies, Waldbroon, Germany). .. Small RNA-seq libraries were performed according to NEBNext® Multiplex Small RNA Library Prep Set for Illumina instructions with a different bar code for each sample (#E7300S, New England Biolabs, Inc.).

    RNA Extraction:

    Article Title: A comprehensive profile of circulating RNAs in human serum
    Article Snippet: Glycogen (Cat. no AM9510, Invitrogen) was used as carrier during the RNA extraction step. .. Small RNA-seq was performed using NEBNext® Small RNA Library Prep Set for Illumina (Cat. No E7300, New England Biolabs Inc.).

    Article Title: Circulating small non-coding RNAs associated with age, sex, smoking, body mass and physical activity
    Article Snippet: Glycogen (Cat. no AM9510, Invitrogen) was used as carrier during the RNA extraction step. .. Small RNAseq was performed using NEBNext® Small RNA Library Prep Set for Illumina (Cat. No E7300, New England Biolabs Inc.) with a cut size on the pippin prep (Cat. No CSD3010, Sage Science) covering RNA molecules from 17 to 47 nucleotides.

    Selection:

    Article Title: A comprehensive profile of circulating RNAs in human serum
    Article Snippet: Small RNA-seq was performed using NEBNext® Small RNA Library Prep Set for Illumina (Cat. No E7300, New England Biolabs Inc.). .. Size selection was performed using a 3% Agarose Gel Cassette (Cat. No CSD3010) on a Pippin Prep (Sage Science) with a cut size optimized to cover RNA molecules from 17 to 47 nt in length.

    Sample Prep:

    Article Title: Chromatin Profiles of Chromosomally Integrated Human Herpesvirus-6A
    Article Snippet: .. MicroRNA (miRNA) libraries were prepared using NEBNext Multiplex Small RNA Sample Prep for Illumina (#E7300) following the manufacturer’s protocol. .. Library quantification and quality were assessed as described above, and pooled libraries were sequenced on an Illumina HiSeq 2,500 at the Genomics Core Facility (University of Texas Health Science Center, San Antonio, TX) to obtain 50-bp single-end reads. miRNA data were processed using the Oasis2 package ( ).

    Ethanol Precipitation:

    Article Title: FIERY1 promotes microRNA accumulation by suppressing rRNA-derived small interfering RNAs in Arabidopsis
    Article Snippet: .. sRNA library construction and sequencing Total RNA (20 μg) from WT (Col-0), fry1-6 , and fry1-8 was resolved on a 15% urea-PAGE gel, and the sRNA fraction (15–40 nt) was excised. sRNAs in the excised gel were recovered in 0.4 M NaCl, followed by ethanol precipitation. sRNA libraries were constructed using the NEBNext Small RNA Library Prep Set for Illumina (NEB, E7300S) according to the manufacturer’s instructions. .. The libraries were pooled and sequenced to generate 75-bp single-end reads on an Illumina NextSeq CN500 platform at Berry Genomics (Beijing, China).

    Spectrophotometry:

    Article Title: Identification of virus-encoded microRNAs in divergent Papillomaviruses
    Article Snippet: Recovered RNA was dissolved in nuclease free water (Ambion) and quantitated with a NanoDrop spectrophotometer (ThermoFisher). .. Libraries were prepared using 180 ng of pooled RNA with the NEBNext Multiplex Small RNA Library Prep Set for Illumina (E7300, New England Biolabs) according to the manufacturer’s instructions with the addition of an additional round of indexing PCR to compensate for low input RNA.

    Immunoprecipitation:

    Article Title: The MID-PIWI module of Piwi proteins specifies nucleotide- and strand-biases of piRNAs
    Article Snippet: Nucleic acids were isolated from immunoprecipitated HA-tagged protein complexes and resolved by 15% urea-PAGE. .. After purification by phenol-chloroform extraction, deep sequencing libraries were prepared using NEBNext Multiplex Small RNA Library Prep Set for Illumina (Cat. No. E7300) according to the manufacturer's instructions.

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    New England Biolabs small rna library prep
    No evidence for an RNAi suppressive function of CVB3 3A protein. ( A ) Schematic representation of the CVB3 genome with an alignment of the 3A sequence with the HEV71 3A sequence, highlighting mutations introduced into CVB3 3A. ( B ) Viral titers of passage 0 (p0) and passage 1 (p1) CVB3 virus stocks harvested from HeLa cells. Viral titers were determined by end-point serial dilution on HeLa cells. ( C ) Top panels, size profiles of small <t>RNAs</t> mapping to the positive viral <t>RNA</t> strand (red) or negative RNA strand (blue) of CVB3. Reads were mapped allowing one mismatch and normalized to the total library size and to viral RNA levels in the sample used to prepare the sequencing libraries (relative to CVB3 WT infection in each cell line). Lower panels, distribution of 21–23 nt viral small RNAs across the CVB3 genome. 5′ positions of normalized reads are plotted. ( D ) Viral titers of supernatant harvested from CVB3 WT and CVB3 D24A/K41Q infected WT or AGO2 KO HeLa and HEK293T cells at 8, 16 and 24 hpi with p0 virus. Bars represent means and SD of 3 biological replicates.
    Small Rna Library Prep, supplied by New England Biolabs, used in various techniques. Bioz Stars score: 95/100, based on 297 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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    No evidence for an RNAi suppressive function of CVB3 3A protein. ( A ) Schematic representation of the CVB3 genome with an alignment of the 3A sequence with the HEV71 3A sequence, highlighting mutations introduced into CVB3 3A. ( B ) Viral titers of passage 0 (p0) and passage 1 (p1) CVB3 virus stocks harvested from HeLa cells. Viral titers were determined by end-point serial dilution on HeLa cells. ( C ) Top panels, size profiles of small RNAs mapping to the positive viral RNA strand (red) or negative RNA strand (blue) of CVB3. Reads were mapped allowing one mismatch and normalized to the total library size and to viral RNA levels in the sample used to prepare the sequencing libraries (relative to CVB3 WT infection in each cell line). Lower panels, distribution of 21–23 nt viral small RNAs across the CVB3 genome. 5′ positions of normalized reads are plotted. ( D ) Viral titers of supernatant harvested from CVB3 WT and CVB3 D24A/K41Q infected WT or AGO2 KO HeLa and HEK293T cells at 8, 16 and 24 hpi with p0 virus. Bars represent means and SD of 3 biological replicates.

    Journal: Scientific Reports

    Article Title: No evidence for viral small RNA production and antiviral function of Argonaute 2 in human cells

    doi: 10.1038/s41598-019-50287-w

    Figure Lengend Snippet: No evidence for an RNAi suppressive function of CVB3 3A protein. ( A ) Schematic representation of the CVB3 genome with an alignment of the 3A sequence with the HEV71 3A sequence, highlighting mutations introduced into CVB3 3A. ( B ) Viral titers of passage 0 (p0) and passage 1 (p1) CVB3 virus stocks harvested from HeLa cells. Viral titers were determined by end-point serial dilution on HeLa cells. ( C ) Top panels, size profiles of small RNAs mapping to the positive viral RNA strand (red) or negative RNA strand (blue) of CVB3. Reads were mapped allowing one mismatch and normalized to the total library size and to viral RNA levels in the sample used to prepare the sequencing libraries (relative to CVB3 WT infection in each cell line). Lower panels, distribution of 21–23 nt viral small RNAs across the CVB3 genome. 5′ positions of normalized reads are plotted. ( D ) Viral titers of supernatant harvested from CVB3 WT and CVB3 D24A/K41Q infected WT or AGO2 KO HeLa and HEK293T cells at 8, 16 and 24 hpi with p0 virus. Bars represent means and SD of 3 biological replicates.

    Article Snippet: 5 µl of the purified small RNAs was used as input for library preparation using the NEBNext Small RNA Library Prep (NEB E7300S/E7580S).

    Techniques: Sequencing, Serial Dilution, Infection

    Increased piRNA sizes and reduced normal piRNAs in adult Pnldc1 Mut testes. a piRNA extension in Pnldc1 Mut mice. Total RNAs from adult WT and Pnldc1 Mut (Mut-1 and Mut-2) testes were end-labeled with [ 32 P]-ATP, and detected by 15% TBE urea gel and autoradiography. Square bracket indicates extended piRNAs. b MILI-piRNA extension in Pnldc1 Mut (Mut-1) mice. Small RNAs were isolated from immunoprecipitated MILI RNPs and were end-labeled with [ 32 P]-ATP, and detected by 15% TBE urea gel and autoradiography. Western blotting was performed with anti-MILI antibody to show immunoprecipitation efficiency. Square bracket indicates extended piRNAs. M molecular weight marker. c MIWI-piRNA extension and reduction in Pnldc1 Mut (Mut-1) mice. Small RNAs were isolated from immunoprecipitated MIWI RNPs and were end-labeled with [ 32 P]-ATP, and detected by 15% TBE urea gel and autoradiography. Western blotting was performed with anti-MIWI antibody to show immunoprecipitation efficiency. Square bracket indicates extended piRNAs. M molecular weight marker. d The length distribution of small RNAs from adult WT and Pnldc1 Mut (Mut-1 and Mut-2) testicular small RNA libraries. Data were normalized by microRNA reads (21–23 nt). e The length distribution of MILI-piRNAs from adult WT and Pnldc1 Mut (Mut-1 and Mut-2) MILI-piRNA libraries. f The length distribution of MIWI-piRNAs from adult WT and Pnldc1 Mut (Mut-1 and Mut-2) MIWI-piRNA libraries

    Journal: Nature Communications

    Article Title: PNLDC1 is essential for piRNA 3′ end trimming and transposon silencing during spermatogenesis in mice

    doi: 10.1038/s41467-017-00854-4

    Figure Lengend Snippet: Increased piRNA sizes and reduced normal piRNAs in adult Pnldc1 Mut testes. a piRNA extension in Pnldc1 Mut mice. Total RNAs from adult WT and Pnldc1 Mut (Mut-1 and Mut-2) testes were end-labeled with [ 32 P]-ATP, and detected by 15% TBE urea gel and autoradiography. Square bracket indicates extended piRNAs. b MILI-piRNA extension in Pnldc1 Mut (Mut-1) mice. Small RNAs were isolated from immunoprecipitated MILI RNPs and were end-labeled with [ 32 P]-ATP, and detected by 15% TBE urea gel and autoradiography. Western blotting was performed with anti-MILI antibody to show immunoprecipitation efficiency. Square bracket indicates extended piRNAs. M molecular weight marker. c MIWI-piRNA extension and reduction in Pnldc1 Mut (Mut-1) mice. Small RNAs were isolated from immunoprecipitated MIWI RNPs and were end-labeled with [ 32 P]-ATP, and detected by 15% TBE urea gel and autoradiography. Western blotting was performed with anti-MIWI antibody to show immunoprecipitation efficiency. Square bracket indicates extended piRNAs. M molecular weight marker. d The length distribution of small RNAs from adult WT and Pnldc1 Mut (Mut-1 and Mut-2) testicular small RNA libraries. Data were normalized by microRNA reads (21–23 nt). e The length distribution of MILI-piRNAs from adult WT and Pnldc1 Mut (Mut-1 and Mut-2) MILI-piRNA libraries. f The length distribution of MIWI-piRNAs from adult WT and Pnldc1 Mut (Mut-1 and Mut-2) MIWI-piRNA libraries

    Article Snippet: Small RNA libraries and bioinformatics Small RNA libraries from immunoprecipitated RNAs or total RNA were prepared using NEBNext Multiplex Small RNA Library Prep Kit (E7300, NEB) following manufacturer’s instructions.

    Techniques: Mouse Assay, Labeling, Autoradiography, Isolation, Immunoprecipitation, Western Blot, Molecular Weight, Marker

    Increased piRNA lengths and reduced piRNA levels in adult Tdrkh cKO testes. ( A ) Severe reduction of total piRNAs in Tdrkh cKO testes. Total RNA from adult WT and Tdrkh cKO testes were end-labeled with [ 32 P]-ATP and detected by 15% TBE urea gel and autoradiography. ( B ) The length distribution of small RNAs from adult WT and Tdrkh cKO testicular small RNA libraries. Data were normalized by miRNA reads (21–23nt). ( C ) Extension of MILI-piRNAs in Tdrkh cKO testes. Small RNAs were isolated from immunoprecipitated MILI-RNPs, end-labeled with [ 32 P]-ATP and detected by 15% TBE urea gel and autoradiography. Western blotting was performed using anti-MILI antibody to show immunoprecipitation efficiency. Square bracket indicates extended piRNAs. M: molecular weight marker. ( D ) Diminished MIWI-piRNAs in Tdrkh cKO testes. Small RNAs were isolated from immunoprecipitated MIWI-RNPs, end-labeled with [ 32 P]-ATP and detected by 15% TBE urea gel and autoradiography. Western blotting was performed using anti-MIWI antibody to show immunoprecipitation efficiency. M: molecular weight marker. ( E ) The length distribution of MILI-piRNAs from adult WT and Tdrkh cKO MILI-piRNA libraries. ( F ) The length distribution of MIWI-piRNAs from adult WT and Tdrkh cKO MIWI-piRNA libraries.

    Journal: Nucleic Acids Research

    Article Title: Mitochondrial membrane-based initial separation of MIWI and MILI functions during pachytene piRNA biogenesis

    doi: 10.1093/nar/gky1281

    Figure Lengend Snippet: Increased piRNA lengths and reduced piRNA levels in adult Tdrkh cKO testes. ( A ) Severe reduction of total piRNAs in Tdrkh cKO testes. Total RNA from adult WT and Tdrkh cKO testes were end-labeled with [ 32 P]-ATP and detected by 15% TBE urea gel and autoradiography. ( B ) The length distribution of small RNAs from adult WT and Tdrkh cKO testicular small RNA libraries. Data were normalized by miRNA reads (21–23nt). ( C ) Extension of MILI-piRNAs in Tdrkh cKO testes. Small RNAs were isolated from immunoprecipitated MILI-RNPs, end-labeled with [ 32 P]-ATP and detected by 15% TBE urea gel and autoradiography. Western blotting was performed using anti-MILI antibody to show immunoprecipitation efficiency. Square bracket indicates extended piRNAs. M: molecular weight marker. ( D ) Diminished MIWI-piRNAs in Tdrkh cKO testes. Small RNAs were isolated from immunoprecipitated MIWI-RNPs, end-labeled with [ 32 P]-ATP and detected by 15% TBE urea gel and autoradiography. Western blotting was performed using anti-MIWI antibody to show immunoprecipitation efficiency. M: molecular weight marker. ( E ) The length distribution of MILI-piRNAs from adult WT and Tdrkh cKO MILI-piRNA libraries. ( F ) The length distribution of MIWI-piRNAs from adult WT and Tdrkh cKO MIWI-piRNA libraries.

    Article Snippet: Small RNA libraries and bioinformatics Small RNA libraries from immunoprecipitated RNAs or total RNA were prepared using NEBNext® Multiplex Small RNA Library Prep Kit (E7300, NEB) following manufacturer's instructions.

    Techniques: Labeling, Autoradiography, Isolation, Immunoprecipitation, Western Blot, Molecular Weight, Marker

    Overview of cross-linking immunoprecipitation with high-throughput sequencing (CLIP-seq) analysis of Pseudomonas aeruginosa Hfq under planktonic and biofilm conditions. (A) Autoradiogram and Western blotting of the CLIP-enriched Hfq-RNA complex under two physiological conditions. XL+, cross-linking; XL-, non-cross-linking; P, planktonic; B, biofilm. Biological replicates are shown in Fig. S1 in the supplemental material. (B) The distribution of Hfq peaks throughout the P. aeruginosa PAO1 genome. Peaks from planktonic and biofilm conditions are highlighted in blue and red, respectively. (C) The Venn diagram shows the number of detected peaks under planktonic and biofilm conditions. Two peaks from both conditions wherein both the start and stop positions are within 40 nt are regarded as the same peak. (D) Classification of Hfq peaks into RNA classes (5′ UTR, CDS, 3′ UTR, sRNA, tRNA, and intergenic peaks). The 5′ UTRs and 3′ UTRs were annotated from TSSs validated by differential RNA-seq ( 39 ) and terminators predicted by TransTermHP ( 40 ), as well as manual curation of sRNAs from size selection sRNA-seq conducted by previous researches ( 16 , 17 ). (E) DAVID enrichment analysis of Hfq peaks from 502 and 180 mRNAs except for intergenic regions under planktonic and biofilm conditions, respectively. The results of KEGG pathway enrichment analysis are presented. Overall results are shown in Table S2 .

    Journal: mSystems

    Article Title: Conditional Hfq Association with Small Noncoding RNAs in Pseudomonas aeruginosa Revealed through Comparative UV Cross-Linking Immunoprecipitation Followed by High-Throughput Sequencing

    doi: 10.1128/mSystems.00590-19

    Figure Lengend Snippet: Overview of cross-linking immunoprecipitation with high-throughput sequencing (CLIP-seq) analysis of Pseudomonas aeruginosa Hfq under planktonic and biofilm conditions. (A) Autoradiogram and Western blotting of the CLIP-enriched Hfq-RNA complex under two physiological conditions. XL+, cross-linking; XL-, non-cross-linking; P, planktonic; B, biofilm. Biological replicates are shown in Fig. S1 in the supplemental material. (B) The distribution of Hfq peaks throughout the P. aeruginosa PAO1 genome. Peaks from planktonic and biofilm conditions are highlighted in blue and red, respectively. (C) The Venn diagram shows the number of detected peaks under planktonic and biofilm conditions. Two peaks from both conditions wherein both the start and stop positions are within 40 nt are regarded as the same peak. (D) Classification of Hfq peaks into RNA classes (5′ UTR, CDS, 3′ UTR, sRNA, tRNA, and intergenic peaks). The 5′ UTRs and 3′ UTRs were annotated from TSSs validated by differential RNA-seq ( 39 ) and terminators predicted by TransTermHP ( 40 ), as well as manual curation of sRNAs from size selection sRNA-seq conducted by previous researches ( 16 , 17 ). (E) DAVID enrichment analysis of Hfq peaks from 502 and 180 mRNAs except for intergenic regions under planktonic and biofilm conditions, respectively. The results of KEGG pathway enrichment analysis are presented. Overall results are shown in Table S2 .

    Article Snippet: A cDNA library of the CLIP samples was prepared using an NEBNext Multiplex Small RNA Library Prep Set for Illumina (E7300; NEB) in accordance with the manufacturers’ instructions with certain modifications.

    Techniques: Cross-linking Immunoprecipitation, Next-Generation Sequencing, Western Blot, RNA Sequencing Assay, Selection

    Normalization and comparison of cross-linking immunoprecipitation with high-throughput sequencing of Hfq between planktonic and biofilm conditions. (A) A schematic representation of the comparison of RNA affinities to Hfq between planktonic and biofilm forms. Only overlapping peaks between two phenotypes were considered. Read coverages of cross-linked samples from planktonic conditions were divided by those from biofilms. Concomitantly, read coverages of total RNA from the planktonic condition were divided by those from biofilms. To remove the effect of RNA expression from peak information, fold changes of CLIP-normalized read coverages between planktonic and biofilm conditions were divided by those of total RNA-normalized coverage. (B to D) Mean coverages normalized according to size factors of overlapping peaks detected by CLIP-seq (B) and total RNA-seq (C) between planktonic and biofilm conditions. Dashed lines denote the reference diagonal. (D) Affinity comparison of Hfq normalized peaks on overlapping RNAs between planktonic and biofilm conditions. Dashed lines denote the reference bases. Peaks from sRNAs and others are highlighted in red and gray, respectively. Significantly enriched sRNAs in planktonic (PhrS) or biofilm (PrrF1/2) cultures are indicated with circles. The correlation coefficient ρ was calculated from planktonic versus biofilm.

    Journal: mSystems

    Article Title: Conditional Hfq Association with Small Noncoding RNAs in Pseudomonas aeruginosa Revealed through Comparative UV Cross-Linking Immunoprecipitation Followed by High-Throughput Sequencing

    doi: 10.1128/mSystems.00590-19

    Figure Lengend Snippet: Normalization and comparison of cross-linking immunoprecipitation with high-throughput sequencing of Hfq between planktonic and biofilm conditions. (A) A schematic representation of the comparison of RNA affinities to Hfq between planktonic and biofilm forms. Only overlapping peaks between two phenotypes were considered. Read coverages of cross-linked samples from planktonic conditions were divided by those from biofilms. Concomitantly, read coverages of total RNA from the planktonic condition were divided by those from biofilms. To remove the effect of RNA expression from peak information, fold changes of CLIP-normalized read coverages between planktonic and biofilm conditions were divided by those of total RNA-normalized coverage. (B to D) Mean coverages normalized according to size factors of overlapping peaks detected by CLIP-seq (B) and total RNA-seq (C) between planktonic and biofilm conditions. Dashed lines denote the reference diagonal. (D) Affinity comparison of Hfq normalized peaks on overlapping RNAs between planktonic and biofilm conditions. Dashed lines denote the reference bases. Peaks from sRNAs and others are highlighted in red and gray, respectively. Significantly enriched sRNAs in planktonic (PhrS) or biofilm (PrrF1/2) cultures are indicated with circles. The correlation coefficient ρ was calculated from planktonic versus biofilm.

    Article Snippet: A cDNA library of the CLIP samples was prepared using an NEBNext Multiplex Small RNA Library Prep Set for Illumina (E7300; NEB) in accordance with the manufacturers’ instructions with certain modifications.

    Techniques: Cross-linking Immunoprecipitation, Next-Generation Sequencing, RNA Expression, RNA Sequencing Assay