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| Status |
Public on Mar 08, 2022 |
| Title |
WT_DMSO_rep1 |
| Sample type |
SRA |
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| Source name |
HCT116 cells
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HCT116
genotype: wild-type
treatment: DMSO (mock)
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| Treatment protocol |
1x10^7 WT or COF-AID tagged cells per replicate were treated as follows: 1. Control: water (mock) treatment for 3h [MED14- and BRD4-AID-tagged cells] or 12h [MED14-AID-tagged cells], or DMSO treatment for 3h [WT] 2. Nutlin: 10µM Nutlin 3a (Sigma# SML0580) treatment for 3h [WT, MED14- and BRD4-AID-tagged cells] or 6h [MED14-AID-tagged cells] 3. Auxin: 500µM auxin (IAA) treatment for 3h [MED14- and BRD4-AID-tagged cells] or 12h [MED14-AID-tagged cells] 4. Auxin + Nutlin: 500µM auxin (IAA) treatment for 3h and subsequent 10µM Nutlin 3a treatment for 3h [MED14- and BRD4-AID-tagged cells] or 500µM auxin (IAA) treatment for 12h and subsequent 10µM Nutlin 3a treatment for 6h [MED14-AID-tagged cells]
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| Extracted molecule |
nuclear RNA |
| Extraction protocol |
After treatment the cells were harvested and nuclei were isolated. Spike-in control (D. melanogaster S2 cell nuclei; 1% of total human HCT116 cells/nuclei) were added at the level of nuclei permeabilization step. Nuclear-run-on was performed for 3min at 37°C with biotin labeled CTPs (Perkin Elmer #NEL542001EA) followed by RNA extraction and base hydrolysis. Biotin-tagged nuclear-run-on RNA was enriched via M280 streptavidin beads (Invitrogen #112.06D) and precipitated via Phenol-Chloroform treatment.
3’ RNA adapters were ligated to isolated RNA and second biotin RNA enrichment followed by RNA 5’ cap modification via TAP (Biozym #187005) treatment was performed. Furthermore, 5’ hydroxyl repair via PNK (NEB #M0201S) treatment and subsequent 5’ adapter ligation was carried out. Afterwards, cDNA was generated from enriched RNA via reverse transcription (Super Script III Reverse Transcriptase, Invitrogen #18080-044). 10µl of the cDNA library was amplified via KAPA Amplification reaction (Roche #7959028001) on a qPCR machine (Biorad CFX Connect RealTime System). KAPA reaction: 10µl cDNA, 1µl forward primer 35µM (RP1-RP20), 1µl of reverse primer 35µM (RP1: 5’-AATGATACGGCGACCACCGAGATCTACAGTTCAGAGTTCTACAGTCCGA-3’), 25µl 2x KAPA SYBER master mix, 13µl water. PCR program: 98°C - 45 sec, 98°C - 15 sec, 60°C - 30 sec, 72°C - 30 sec, 72°C - 10 sec. Samples were removed from the qPCR machine after 12-15 cycles and cleaned with Ampure beads (Beckman #A63881) in a 1:1.4 (DNA:beads) ratio. DNA bound to the beads was eluted in 11µl of water and subjected to single-end deep sequencing.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 550 |
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| Description |
WT_DMSO.ps.bw
WT_DMSO.ns.bw
WT_3hrNutlin_raw_counts_per_condition_for_promoter_and_genebody_regions.txt
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| Data processing |
Library strategy: PRO-seq
UMI removal and adapter trimming: Sequenced reads consist of a 10nt long UMI (for counting unique RNA molecules), followed by PolII active site-associated RNA sequence and potentially a part of the Illumina sequencing adapter (in cases where RNA was shorter than 22nt). Prior to mapping, the UMI sequence (10nt at the 5' end of the read) was removed and noted for later counting. Illumina adapter was trimmed off with cutadapt v1.18.
Read alignment: All reads longer than 15nt after adapter trimming were mapped to a merged reference consisting of hg19 and dm3 (spike-in) genome using Bowtie version 0.12.9 allowing 2 mis-matches. Reads mapping to multiple positions were randomly assigned to one mapping position.
Genomic coverage: 5' ends of mapped reads (the last nucleotide transcribed by PolII) were counted per genomic position taking into account only unique UMIs. Coverage was generated separately for positive and negative strands. Coverage was normalized to tags per million and scaled by a factor obtained from relative spike-in abundance. Coverage was generated with GenomicRanges v1.34.0 package in R and exported into bigWig format with rtracklayer v1.42.2.
Quantification and differential analysis: 5' ends of mapped, UMI-collapsed reads overlapping promoter+pause region (from +1 to +150bp downstream of the TSS) and gene body region (from +150bp to annotated gene end) were counted for each individual sample/replicate. Gene annotation from Ensembl v83 was used, and annotated TSSs were corrected to nearest CAGE-defined TSS from FANTOM5 collection. Provided raw counts were used for downstream differential analysis between different conditions with DEseq2 v1.22.2.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text files include genomic coordinates of a referent set of 21115 genes/transcripts with Ensembl v83 gene/transcript IDs, gene names, TSS positions corrected by CAGE and annotated transcription end sites. For each gene raw counts in each individual replicate are provided, separately for promoter+pause region (from +1 to +150bp downstream of the TSS) and gene body region (from +150bp to annotated gene end). BigWig files contain spike-in normalized genome-wide coverage of PRO-seq reads, separately for positive and negative strands (the values for negative strand are shown as negative).
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| Submission date |
Sep 15, 2021 |
| Last update date |
Mar 08, 2022 |
| Contact name |
Vanja Haberle |
| E-mail(s) |
vanja.haberle@imp.ac.at
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| Organization name |
The Research Institute of Molecular Pathology (IMP)
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| Lab |
Stark Lab
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| Street address |
Campus-Vienna-Biocenter 1
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| City |
Vienna |
| ZIP/Postal code |
1030 |
| Country |
Austria |
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| Platform ID |
GPL21697 |
| Series (2) |
| GSE156737 |
Differential cofactor dependencies define functionally distinct types of human enhancers (PRO-seq) |
| GSE156741 |
Differential cofactor dependencies define functionally distinct types of human enhancers |
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| Relations |
| BioSample |
SAMN21444918 |
| SRA |
SRX12196958 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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