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| Status |
Public on Dec 11, 2015 |
| Title |
Lib19 |
| Sample type |
SRA |
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| Source name |
THP1_WT_ChIP-seq
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| Organism |
Homo sapiens |
| Characteristics |
cell line: THP1
cell type: human acute monocytic leukemia (AML) cell line
genotype/variation: wild type (control)
chip antibody: CDC73
chip antibody vendor: Bethyl
chip antibody cat. #: A300-170A
fixation: EGS+ FA
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| Treatment protocol |
For knockdown experiments with lentiviral shRNAs, TRC control or gene-specific shRNA was transduced into cells by spin infection. Around 15 hours after transduction, cells were washed with PBS twice, and re-suspended in fresh medium. Puromycin (2 μg/ml, final conc.) was added to the culture medium 24 hours later. Cells were harvested for ChIP or RNA extraction 3 days after puromycin selection. For flavopiridol (Sigma, cat. no. F3055) treatment of THP1 cells, flavopiridol was solubilized in DMSO and added to culture medium at 1 μM (final conc.). Meanwhile, an equal volume of DMSO was added to control cells. After 1 more hour in culture, cells were fixed for ChIP.
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| Growth protocol |
The human AML cell line THP1 and the human ALL cell line CCRF-CEM were grown in RPMI-160 medium supplemented with 10% FBS and 2% Penicillin/Streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP assays, normally, cells were fixed with 0.4% (v/v) formaldehyde at room temperature for 10 min. For improving the ChIP efficiency of non-DNA binding factors, double fixation was used. For double fixation with EGS (Pierce, Cat. no. 21565) and formaldehyde, cells were fixed initially with 1.5 mM EGS at room temperature for 30 min, and subsequently with 0.4% formaldehyde at room temperature for 10 min. For double fixation with DMA (Pierce, Cat. no. 20660) and formaldehyde, cells were fixed initially with 25 mM DMA at room temperature for 1 hour, and subsequently with 0.4% formaldehyde at room temperature for 10 min. After two washes with PBS, fixed cells were resuspended in ice-cold RIPA-0.3 buffer (10 mM Tris-HCl, 1mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% NaDOC, and 0.3 M NaCl, pH7.4) at the concentration of 40 million/ml, and chromatin was disrupted by sonication to the size range of 100 to 500 bp. Antibodies were diluted in RIPA-0.3, and bound to Dynabeads protein A (Life Technologies, cat. no. 10002D) by incubating at 4 °C for 3 hours. The bead-antibody complexes were washed with RIPA-0.3 twice, and then incubated with sonicated chromatin at 4 °C overnight. The second day, after 2 washes with RIPA-0.5, 1 wash with RIPA-0.3, 1 wash with RIPA-0, 2 washes with LiCl buffer (10 mM Tris-HCl, 0.25 M LiCl, 0.25% NP-40, and 0,25% NaDOC, pH7.4), and 2 washes with TE buffer, bound protein-DNA complexes were resuspended in elution buffer (10 mM Tris-HCl, 1mM EDTA, and 1% SDS, pH7.4) supplemented with 10 µg/ml RNase A for elution and RNA digestion, and incubated at 55 °C for 1 hour. Afterwards, proteinase K was added to the final concentration of 100 µg/ml, and 30 min later, the temperature of incubation was increased to 65 °C for decrosslinking. After decrosslinking for 4–6 hours, DNA was purified by ChIP DNA Clean & Concentrator (Zymo Research, cat. no. D5205). RNA was extracted from cells using RNeasy Plus Mini Kit (Qiagen, cat. no. 74134) or Quick-RNA MiniPrep Kit (Zymo, R1054) by following the manufacturer’s protocol (Qiagen, cat. no. 74134).
ChIP-seq libraries were constructed with 5 to 10 ng immunoprecipitated DNA. RNA-seq libraries were prepared by following a previously published strand-specific protocol. Samples were sequenced on HiSeq 2000 or 2500 by following the manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
Illumina Casava1.7 software used for basecalling.
Raw sequenced reads were mapped to reference genome hg19 using BWA with default settings through GALAXY.
ChIPseq peaks were called by SICER software with parameter settings as W=200,G=400, and FDR=0.001
Reads Per Kilobase of exon per Megabase of library size (RPKM) were calculated using rpkmforgenes.py developed by Sandberg.
Genome_build: hg19
Supplementary_files_format_and_content: tab-delimited text files include RPKM values for each RNA-seq Sample; bedGraph files were generated for each ChIP-Seq sample.
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| Submission date |
Oct 08, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Robert Roeder |
| E-mail(s) |
roeder@rockefeller.edu
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| Organization name |
The Rockefeller University
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| Street address |
1230 York Avenue
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| City |
New York |
| State/province |
NY |
| ZIP/Postal code |
10065 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE62171 |
RNA Polymerase II-associated factor 1 regulates the release and phosphorylation of paused RNA Polymerase II |
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| Relations |
| BioSample |
SAMN03099707 |
| SRA |
SRX728769 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1520908_Lib19.bedGraph.gz |
37.3 Mb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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