LN-2683GS cells were treated with 1 µM JQ1, DMSO, and 1000 units/mL IFN-α for 2 hours. U87MG were treated with 1 µM JQ1 or DMSO for 24 hours. Cells were cross-linked and ChIP was performed using iDeal ChIP-seq kit for Transcription Factors (Diagenode) as detailed in the Supplementary Methods , using the following antibodies: anti-Pol II (Cell Signaling, D8L4Y, 1:50), anti-BRD4 (Bethyl Laboratories, A301, 1:60), anti-H3K4me3 (Diagenode, C15410003, 1:300), anti-H3K27ac (Abcam, ab4729, 1:300 or 1:600), control IgG (1:600, Diagenode).
Anti pol 2
Manufactured by Cell Signaling Technology
Anti-Pol II is a laboratory product that detects RNA polymerase II, a critical enzyme involved in the transcription of genetic information from DNA to RNA. It is a highly specific antibody that can be used in various research techniques to study gene expression and regulation.
Automatically generated - may contain errors
Lab products found in correlation
Anti brd4, by Fortis Life Sciences (2 mentions)
Ab4729, by Abcam (1 mentions)
Anti h3k4me3, by Diagenode (1 mentions)
Control igg, by Diagenode (1 mentions)
Anti h3k27ac, by Abcam (1 mentions)
Ideal chip seq kit for transcription factors, by Diagenode (1 mentions)
Rna extraction reagent, by Solarbio (1 mentions)
Anti snrnp70, by Merck Group (1 mentions)
Magnetic bead, by Merck Group (1 mentions)
Anti h3k27me3, by Merck Group (1 mentions)
Anti h3k27ac, by Active Motif (1 mentions)
Anti h3k4me1, by Active Motif (1 mentions)
Anti h3k4me3, by Active Motif (1 mentions)
Anti cdk9, by Santa Cruz Biotechnology (1 mentions)
Nextseq 500, by Illumina (1 mentions)
Ddpcr evagreen supermix, by Bio-Rad (1 mentions)
Qiaquick pcr purification column, by Qiagen (1 mentions)
Anti p300, by Santa Cruz Biotechnology (1 mentions)
3 protocols using anti pol 2
1
Chromatin Profiling of JQ1 Responses
2
RIP Assay for Identifying RNA-Binding Proteins
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RIP assays were performed using a kit from Millipore (Cat. no. 17-701) and following the manufacturer's instructions. Neuro-2a cells (2×10 7 ) were lysed in complete RIP lysis buffer and the cell lysates were divided into equal parts and incubated (overnight with rotation, at 4°C) with anti-Pol II (Cell Signaling Technology (CST), Cat. no. #2629), anti-U1A (Abcam, Cat. no. ab166890), anti-U2AF65 (Abcam, Cat. no. ab37530), IgG (Millipore, Cat. no. pp64B), or anti-SNRNP70 (Millipore, Cat. no. CS203216). Next, magnetic beads (Millipore, Cat. no. CS203178) were added to the cell lysates and the incubation was continued at 4°C for 1 h, after which the samples were incubated with proteinase K at 55°C for 1 h. The enriched RNA was obtained using RNA Extraction Reagent (phenol:chloroform:isoamylol = 125:24:1; pH<5.0; Solar Bio), and qRT-PCR was performed on the purified RNA to detect the targets of interest.
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted July 10, 2020. ; https://doi.org/10.1101/2020.07.08.192716 doi: bioRxiv preprint
(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.
The copyright holder for this preprint this version posted July 10, 2020. ; https://doi.org/10.1101/2020.07.08.192716 doi: bioRxiv preprint
3
ChIP-Seq Protocol for Histone Marks
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Cells were crosslinked in 1% formaldehyde for 15 min and then quenched with 125 mM glycine for 5 min. Cells were lysed and chromatin sheared by sonication to an average length of 300-500 bp. IP was carried out using the following antibodies: anti-H3K27Ac (Active Motif), anti-H3H18Ac (Active Motif), anti-H3K4me1 (Active Motif), anti-H3K4me3 (Active Motif), anti-H3K27me3 (Millipore), anti-P300 (Santa Cruz Biotechnology), anti-BRD4 (Bethyl Labs), anti-CDK9 (Santa Cruz Biotechnology), and anti-Pol II (Cell Signaling Technology).
Complexes were washed, eluted from beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 C, and DNA was purified using QIAQuick PCR purification columns (QIAGEN). PCR was performed using the EvaGreen ddPCR Supermix (Bio-Rad). For Pol II ChIP qPCR primer locations, see Table S3.
For ChIP-seq, subsequent steps were performed at Active Motif. IP reactions were spiked with Drosophila chromatin (Active Motif 53083) and a Drosophila-specific antibody (Active Motif 61686) as a mechanism for normalization. Complexes were processed as described earlier, and DNA was purified by phenol-chloroform extraction. Illumina genomic adapters were ligated, and the resulting DNA libraries were quantified and sequenced on Illumina NextSeq 500, producing single-end 75 nt reads.
Complexes were washed, eluted from beads with SDS buffer, and subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 C, and DNA was purified using QIAQuick PCR purification columns (QIAGEN). PCR was performed using the EvaGreen ddPCR Supermix (Bio-Rad). For Pol II ChIP qPCR primer locations, see Table S3.
For ChIP-seq, subsequent steps were performed at Active Motif. IP reactions were spiked with Drosophila chromatin (Active Motif 53083) and a Drosophila-specific antibody (Active Motif 61686) as a mechanism for normalization. Complexes were processed as described earlier, and DNA was purified by phenol-chloroform extraction. Illumina genomic adapters were ligated, and the resulting DNA libraries were quantified and sequenced on Illumina NextSeq 500, producing single-end 75 nt reads.
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