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Gel cassette 20b

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The Gel cassette 20B is a laboratory equipment component designed for electrophoresis applications. It provides a standardized platform for casting and running agarose or polyacrylamide gels.

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Lab products found in correlation

Nextseq 500 system, by Illumina (4 mentions) Biotin 11 atp gtp ctp utp, by PerkinElmer (4 mentions) Dynabeads m 280 streptavidin, by Thermo Fisher Scientific (4 mentions) Dynabeads, by New England Biolabs (1 mentions) Phusion hotstart, by Thermo Fisher Scientific (1 mentions) Superscript 3, by Thermo Fisher Scientific (1 mentions) Dynabead, by Thermo Fisher Scientific (1 mentions)

4 protocols using gel cassette 20b

1

PRO-seq Analysis of Nascent Transcription

Cited 1 time
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PRO-seq experiments were performed as described previously (Mahat et al., 2016 (link)). Nuclei were isolated by Dounce homogenizer with loose pestle. 1×10^7 nuclei were subjected to nuclear run-on (30°C, 3 min) in the presence of 25 μM Biotin-11-ATP/GTP/CTP/UTP (PerkinElmer). Total RNA was extracted and hydrolyzed in 0.2 M NaOH (on ice, 10 min). Biotinylated nascent RNAs were purified by Dynabeads M-280 streptavidin (Invitrogen). Following adaptor ligation, cDNA synthesis, and PCR amplification 140–350 bp libraries were size-selected by Pippin HT with 2% gel cassette 20B (Sage Science) then sequenced by NextSeq 500 system (Illumina) with single-read runs. Raw fastq data were trimmed by Cutadapt 1.14 (Martin, 2011 ) and Trimmomatic v0.32 (Bolger et al., 2014 (link)), and then aligned on hg19 or dm3 genome by bowtie 1.1.2 (Langmead et al., 2009 (link)). Strand-specific single nucleotide ends of aligned reads were generated by BEDTools v2.28 with genomecov (Quinlan and Hall, 2010 (link)) as bedgraph format. Bedgraph data were normalized by the number of reads mapped to spike-in dm3 genome, and then converted to bigwig data, which were used for downstream analyses.
Beckedorff F., Blumenthal E., daSilva L.F., Aoi Y., Cingaram P.R., Yue J., Zhang A., Dokaneheifard S., Valencia M.G., Gaidosh G., Shilatifard A, & Shiekhattar R. (2020). The Human Integrator Complex Facilitates Transcriptional Elongation by Endonucleolytic Cleavage of Nascent Transcripts. Cell reports, 32(3), 107917.
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2

PRO-seq Library Preparation Protocol

Cited 2 times
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PRO-seq experiments were performed and analyzed as described previously (28 (link)). Briefly, PRO-seq experiments were performed as described previously (48 (link)). Nuclei were isolated with a Dounce homogenizer with loose pestle. Nuclei (1 × 107) were subjected to nuclear run-on (30°C, 3 min) in the presence of 25 mM Biotin-11-ATP/GTP/CTP/UTP (PerkinElmer). Total RNA was extracted and hydrolyzed in 0.2 M NaOH (on ice, 10 min). Biotinylated nascent RNAs were purified by Dynabeads M-280 streptavidin (Invitrogen). Following adaptor ligation, cDNA synthesis, and PCR amplification, 140– to 350–base pair (bp) libraries were size-selected by Pippin HT with 2% gel cassette 20B (Sage Science) and then sequenced using a NextSeq 500 system (Illumina) with single-read runs. Raw fastq data were trimmed by Cutadapt 1.14 (49 ) and Trimmomatic v0.32 (50 (link)), and then aligned on hg19 or dm3 genome by bowtie 1.1.2 (51 (link)). Strand-specific single-nucleotide ends of aligned reads were generated by BEDTools v2.28 with genomecov (52 (link)) as bedgraph format. Bedgraph data were normalized by the number of reads mapped to spike-in dm3 genome and then converted to bigwig data, which were used for downstream analyses.
Dasilva L.F., Blumenthal E., Beckedorff F., Cingaram P.R., Gomes Dos Santos H., Edupuganti R.R., Zhang A., Dokaneheifard S., Aoi Y., Yue J., Kirstein N., Tayari M.M., Shilatifard A, & Shiekhattar R. (2021). Integrator enforces the fidelity of transcriptional termination at protein-coding genes. Science Advances, 7(45), eabe3393.
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3

PRO-seq Analysis of Nascent Transcription

Cited 1 time
Check if the same lab product or an alternative is used in the 5 most similar protocols
PRO-seq experiments were performed as described previously (Mahat et al., 2016 (link)). Nuclei were isolated by Dounce homogenizer with loose pestle. 1×10^7 nuclei were subjected to nuclear run-on (30°C, 3 min) in the presence of 25 μM Biotin-11-ATP/GTP/CTP/UTP (PerkinElmer). Total RNA was extracted and hydrolyzed in 0.2 M NaOH (on ice, 10 min). Biotinylated nascent RNAs were purified by Dynabeads M-280 streptavidin (Invitrogen). Following adaptor ligation, cDNA synthesis, and PCR amplification 140–350 bp libraries were size-selected by Pippin HT with 2% gel cassette 20B (Sage Science) then sequenced by NextSeq 500 system (Illumina) with single-read runs. Raw fastq data were trimmed by Cutadapt 1.14 (Martin, 2011 ) and Trimmomatic v0.32 (Bolger et al., 2014 (link)), and then aligned on hg19 or dm3 genome by bowtie 1.1.2 (Langmead et al., 2009 (link)). Strand-specific single nucleotide ends of aligned reads were generated by BEDTools v2.28 with genomecov (Quinlan and Hall, 2010 (link)) as bedgraph format. Bedgraph data were normalized by the number of reads mapped to spike-in dm3 genome, and then converted to bigwig data, which were used for downstream analyses.
Beckedorff F., Blumenthal E., daSilva L.F., Aoi Y., Cingaram P.R., Yue J., Zhang A., Dokaneheifard S., Valencia M.G., Gaidosh G., Shilatifard A, & Shiekhattar R. (2020). The Human Integrator Complex Facilitates Transcriptional Elongation by Endonucleolytic Cleavage of Nascent Transcripts. Cell reports, 32(3), 107917.
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4

PRO-seq protocol with modifications

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We performed PRO-seq following a previously described protocol50 (link) with some modifications. Nuclei were isolated by Dounce homogenizer with loose pestle. Approximately 107 nuclei were subjected to nuclear run-on (30 °C, 3 min) in the presence of 25 μM biotin-11-ATP/GTP/CTP/UTP (PerkinElmer) and 6 × 105Drosophila S2 spike-in nuclei. Total RNAs were extracted and hydrolyzed in 0.2 M NaOH (on ice, 10 min). Biotinylated nascent RNA was purified by Dynabeads M-280 streptavidin (Invitrogen). After 3′ VRA3 adapter ligation and the second purification by Dynabeads, 5′ cap and 5′ hydroxyl RNA were converted to 5′ monophosphorylated RNA by RppH (NEB) and PNK (NEB), respectively. After 5′ VRA5 adapter ligation and the third purification by Dynabeads, complementary DNA was generated by reverse transcription with SuperScript III (Invitrogen) and RP1 primer. PCR by Phusion Hot Start (Thermo) and RP1/RPIx primer sets in 10 cycles amplified indexed DNA libraries. DNA libraries of 140–350 bp were size-selected by Pippin HT with 2% gel cassette 20B (Sage Science). Two independent cell cultures per condition were then sequenced by NextSeq 500 system (Illumina) with single-read runs.
Douillet D., Sze C.C., Ryan C., Piunti A., Shah A.P., Ugarenko M., Marshall S.A., Rendleman E.J., Zha D., Helmin K.A., Zhao Z., Cao K., Morgan M.A., Singer B.D., Bartom E., Smith E.R, & Shilatifard A. (2020). Uncoupling histone H3K4 trimethylation from developmental gene expression via an equilibrium of COMPASS, Polycomb, and DNA methylation. Nature genetics, 52(6), 615-625.
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