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| Status |
Public on Jan 18, 2023 |
| Title |
CDK9_RBBP5FKBP_0h |
| Sample type |
SRA |
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| Source name |
mESCs
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| Organism |
Mus musculus |
| Characteristics |
cell type: mESCs
strain: 129/Ola
genotype: RBBP5-FKBP degron
time: 0h
antibody: CDK9
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| Treatment protocol |
Auxin or dTAG-13 was added at the indicated time point.
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| Growth protocol |
E14 ESCs (129/Ola background) were maintained on 0.2% gelatin-coated plates in Glasgow Minimum Essential Medium (GMEM, Sigma-Aldrich, G5154) containing 15% fetal bovine serum (Gibco, 26140079), supplemented with 1× Pen-Strep (Thermofisher Scientific, 15140122), 2 mM Glutamax (Thermofisher Scientific, 35050061), 50 µM β-mercaptoethanol (Thermofisher Scientific, 21985023), 0.1 mM nonessential amino acids (Thermofisher Scientific, 11140050), 1 mM sodium pyruvate (Thermofisher Scientific, 11360070), and Leukemia Inhibitory Factor (LIF, 1000U/mL, Millipore).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ES cells were cross-linked by the addition of 1% formaldehyde (Sigma-Aldrich, 252549-1L) in the dish for 10 minutes at RT before quenching with 0.125 M glycine. The fixed cells were washed with PBS and resuspended in SDS buffer (100 mM NaCl, 50 mM Tris-HCl pH 8.0, 5 mM EDTA, 0.5% SDS, 1x protease inhibitor cocktail from Roche). The resulting nuclei were spin down, resuspended in the immunoprecipitation buffer at 1 mL per 16 million cells (SDS buffer and Triton Dilution buffer (100 mM NaCl, 100 mM Tris-HCl pH 8.0, 5 mM EDTA, 5% Triton X-100) mixed in 2:1 ratio with the addition of 1x protease inhibitor cocktail from Roche) and processed on a Bioruptor Plus Sonicator (Diagenode) to achieve an average fragment length of 200-300 bps.
Libraries were constructed using NEBNext Ultra II DNA Library prep kit (NEB, E7645L).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
NextSeq 550 |
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| Description |
RBBP5-FKBP degron
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| Data processing |
Illumina bcl2fastq2 Conversion Software v2.20 used for basecalling.
FASTQ reads were mapped to the GRCm38 (mm10) genome using Bowtie2 (v2.4.1) using the standard settings.
DeepTools (v3.3.0) was used to generate occupancy heat maps, and the resulting normalized occupancy matrix was used as input for custom R scripts to generate average profile plots and to calculate processivity indices.
Genome_build: mm10
Supplementary_files_format_and_content: All other processed data is made available as supplementary files to the manuscript
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| Submission date |
Aug 11, 2021 |
| Last update date |
Jan 18, 2023 |
| Contact name |
Hua Wang |
| E-mail(s) |
moodswh@gmail.com
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| Organization name |
Memorial Sloan Kettering Cancer Center
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| Street address |
430 East 67th Street
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| City |
New York |
| State/province |
New York |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL21626 |
| Series (2) |
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| Relations |
| BioSample |
SAMN20710415 |
| SRA |
SRX11717265 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5513846_CDK9_RBBP5FKBP_0h.bw |
92.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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