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| Status |
Public on Nov 06, 2024 |
| Title |
shCtrl for PAF1 data Pol II ChIP |
| Sample type |
SRA |
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| Source name |
HEL cell line
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| Organism |
Homo sapiens |
| Characteristics |
treatment: shCtrl
antibody: Pol II
fixation: DMA+FA
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| Treatment protocol |
For double fixation with DMA (Thermo, 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. For sonication, fixed cells were washed twice with PBS and resuspended in ice-cold RIPA-0.3 buffer (10 mM Tris-HCl, 1 mM EDTA, 1% Triton X-100, 0.1% SDS, 0.1% NaDOC, and 0.3 M NaCl, pH 7.4) supplemented with Protease Inhibitor Cocktail (Millipore, cat. no. 535140) at a concentration of 40 million cells/ml; genomic DNA was disrupted to a size range of 100 to 500 bp.
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| Growth protocol |
Human cells HEL were cultured in 90% RPMI-1640 + 10% FBS + 2% Penicillin/Streptomycin.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
For immunoprecipitation, on day 1 antibodies were diluted in RIPA-0.3 and bound to Dynabeads protein A (Thermo, cat. no. 10002D) by incubating at 4 °C for 3 hours. Afterwards, the bead-antibody complexes were washed twice with RIPA-0.3 and then incubated with sonicated chromatin at 4 °C overnight. On day 2, 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, 1 mM EDTA, 0.25 M LiCl, 0.25% NP-40, and 0.25% NaDOC, pH 7.4), and 2 washes with TE buffer, bound protein-DNA complexes were resuspended in elution buffer (10 mM Tris-HCl, 1mM EDTA, 0.2 M NaCl, and 1% SDS, pH 7.4) supplemented with 10 µg/ml RNase A for both elution and RNA digestion, and incubated at 55 °C for 1 hour. Then Proteinase K was added to a final concentration of 200 µg/ml, and after 30 min incubation the temperature was increased to 65 °C for crosslink reversal. After incubation for 4 to 6 hours, DNA was purified by ChIP DNA Clean & Concentrator (Zymo Research, cat. no. D5205).
The libraries were constructed with 2–10 ng immunoprecipitated DNA. After end-repair, A-tailing, and barcode ligation, barcoded DNA was amplified by 16-cycle PCR. Libraries were sequenced on Illumina Genome Analyzer II, HiSeq 2000, or HiSeq 2500.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Raw reads were trimmed for adaptor sequence and low-complexity or low-quality sequence were filtered using fastp v0.13.1 with default parameters. ChIP-seq reads were aligned using bowtie2 v2.3.4.1.
Normalized aboundency in each 50-bp genomic bin were generated into bigWig file using deepTools bamCoverage command with parameter --normalizeUsing CPM.
Genome_build: Hg38
Supplementary_files_format_and_content: BigWig files contains normalized read count (CPM, in 50-bp bins).
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| Submission date |
Nov 08, 2021 |
| Last update date |
Nov 06, 2024 |
| Contact name |
Aiwei Wu |
| E-mail(s) |
aiwei.wu@sjtu.edu.cn
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| Organization name |
Shanghai Jiao Tong University
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| Department |
School of Life Sciences and Biotechnology
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| Lab |
Ming Yu
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| Street address |
800 Dongchuan Road
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| City |
Shanghai |
| State/province |
Shanghai |
| ZIP/Postal code |
200240 |
| Country |
China |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE188413 |
The PAF1 Complex Regulates Transcriptional Termination and Reinitiation [ChIP-seq] |
| GSE188417 |
The PAF1 Complex Regulates Transcriptional Termination and Reinitiation |
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| Relations |
| BioSample |
SAMN22984460 |
| SRA |
SRX13057079 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5681042_HEL_Ctrl.PolII.chip.bw |
130.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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