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| Status |
Public on Jul 30, 2021 |
| Title |
ChIP-Rx Input R2 |
| Sample type |
SRA |
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| Source name |
bone marrow, CML
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| Organism |
Homo sapiens |
| Characteristics |
cell line: K562 dTAG-BRD4
treatment: BRD4 degradation
antibody: no antibody
matched input: --
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| Treatment protocol |
K562 dTAG-BRD4 cells at a concentration of 1 x 10^6/ml were treated with 500 nM dTAG7 or DMSO for 2 h.
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| Growth protocol |
K562 dTAG-BRD4 cells were cultured in RPMI containing 10% FBS, 5% penicillin-streptomycin. NIH3T3 cells, which were used as spike-ins, were grown in DMEM containing 10% FBS , 5% penicillin-streptomycin. K562 dTAG-BRD4 cells were plated at 5 x 10^5 cells/ml, NIH3T3 were plated at 2 x 10^6 cells/T75 flask every two days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
K562 dTAG-BRD4 cells were crosslinked with 1% formaldehyde for 5 min at room temperature, NIH3T3 cells were crosslinked for 8 min. After combining NIH3T3 and K562 dTAG-BRD4 cells in a ratio of 1:5, cells were lysed as described by Baluapuri et al. (2019), followed by sonication using a Covaris E220evolution at intensity 4, duty cycle 5%, 200 cycles per burst for 20 min. Lysates were pre-cleared by centrifugation. Lysates and antibodies coupled to Dynabeads Protein G (Thermo Fisher Scientific) were incubated at 4°C overnight with rotation. Afterwards, the beads were washed with buffers prepared according to Baluapuri et al. (2019). DNA fragments were purified using the ChIP DNA Clean & Concentrator kit (Zymo).
Libraries were constructed from 6 to 30 ng DNA using the NEBNext Ultra II DNA kit (NEB) according to the manufacturer's instructions. After purifying the amplified libraries with AMPure XP beads (Beckman Coulter), fragments of 200 to 500 bp were extracted from an 8% TBE gel. Library quality and quantity were assessed using BioAnalyzer HS DNA (Agilent) and Qubit dsDNA HS (Thermo Fisher) assays, respectively.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
For ChIP-Rx, we aligned the sequencing reads to a joined reference genome which we assembled from human (hg38/GRCh38) and the spiked-in mouse (mm10/GRCm38)
reference genomes using Bowtie2 v2.3.5.1 (Langmead and Salzberg, 2012) in paired-end mode with '-k 1'.
We extracted the raw density from human for the protein of interest using bedtools (Quinlan and Hall, 2010) in the 'genomecov -bg' mode.
Genome_build: Human (hg38/GRCh38) and the spiked-in mouse (mm10/GRCm38)
Supplementary_files_format_and_content: bigWig files were generated using ‘bedGraphToBigWig’; Scores represent Reads per Million (RPM) or the fold-enrichment 'FE' signal over the matched input control.
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| Submission date |
Oct 02, 2020 |
| Last update date |
Jul 30, 2021 |
| Contact name |
Dr. Andreas Mayer |
| Organization name |
Max-Plack-Institute for molecular genetics
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| Street address |
Ihnestraße 63-73
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| City |
Berlin |
| ZIP/Postal code |
14195 |
| Country |
Germany |
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| Platform ID |
GPL24676 |
| Series (2) |
| GSE158965 |
A BRD4-mediated elongation control point primes transcribing RNA polymerase II for 3'-processing and termination [ChIP-seq] |
| GSE158966 |
A BRD4-mediated elongation control point primes transcribing RNA polymerase II for 3'-processing and termination |
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| Relations |
| BioSample |
SAMN16355330 |
| SRA |
SRX9237252 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4816708_MRA94.rpm.bw |
918.7 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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