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| Status |
Public on Sep 29, 2017 |
| Title |
HCT116 H3K27ac-ChIP-seq-WT-Rep1 |
| Sample type |
SRA |
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| Source name |
HCT116
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| Organism |
Homo sapiens |
| Characteristics |
RNAi: scrambled control
antibody source: H3K27ac (Abcam, ab4729)
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| Treatment protocol |
shRNA-mediated gene silencing was performed by lentiviral infection of GIPZ shRNAs (Dharmacon, GE) targeting ARID1B (V3LHS_306691) or scrambled control (RHS4346). 48 hours after infection, puromycin (HCT116: 2μg/ml) was added to select infected cells.
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| Growth protocol |
HCT116 cells were cultured in RPMI (Corning CellGro), 10% FBS (Omega Scientific, Inc), supplemented with 1% Penicllin/Streptavidin (Life Technologies).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ATAC-Seq: Cells were washed with cold PBS, collected by centrifugation then resuspended in resuspension buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2). After collection, cells were lysed in lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% NP-40) and collected before incubating in transposition mix containing Tn5 transposase. ChIP-seq: Cells were harvested and crosslinked in 1% formaldehyde for 10 min before quenching with glycine for 5 min on ice. Cells were pelleted by centrifugation and snap frozen in dry ice before storage at -80˚C. Pellets were thawed on ice and resuspended in rinse buffer 1 (50 mM HEPES pH 8.0, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP40, 0.25% Triton X100), collected by centrifugation then resuspended in rinse buffer 2 (10 mM Tris pH 8.0, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl). Cells were washed and resuspended in shearing buffer before sonication using Covaris E220 (0.1% SDS, 1mM EDTA, pH 8, 10mM Tris HCl, pH 8). For ChIPs using antibodies for histone modifications, 106 cells in 1ml shearing buffer were added to 1ml Covaris tubes and sheared for 12 mins at 140W with 10% duty factor. For FRA1 ChIPs, cells were sheared for 8 mins at 140W with 5% duty factor. DNA was then made up to 1x IP buffer (50 mM HEPES/KOH pH 7.5, 150 mM NaCl , 1 mM EDTA, 1 % Triton X100, 0.1 % DOC, 0.1% SDS, supplemented with protease inhibitors) and 3ug antibody added for overnight incubation with rolling at 4˚C. Antibody bound DNA was recovered using a 1:1 mixture of Protein A and Protein G beads, washed and treated with Proteinase K and RNAse A. Purified ChIP DNA was then used for library generation for ChIP-seq. RNA-Seq: RNA was isolated using Quick-RNA Miniprep Kit (Zymo Research).
ATAC-seq: Purified DNA was ligated with adapters (NuGEN), amplified and size selected for sequencing. Library DNA was sequenced with paired end 42 bp reads. RNA-seq: mRNA was isolated using NEBnext Poly(A) mRNA Magnetic Isolation Module (New England BioLabs) with 5 μg input RNA according to the manufacturer’s guidelines. Libraries were prepared for RNA-seq using NEBnext® UltraTM RNA Library Prep Kit for Illumina® following the manufacturer’s instructions. mRNA was fragmented and purified before first strand and second strand synthesis. Double-stranded cDNA was then purified and ends repaired before dA tailing and adapter ligation. After cleanup and size selection, cDNA libraries were amplified and purified before sequencing with single end 51 bp reads. ChIP-seq: 5 ng ChIP DNA was used to prepare libraries using the NuGen Ovation® Ultralow Library System V2 following the manufacturer’s instructions. Briefly, DNA ends were repaired before ligation of adapters for sequencing. Following bead purification, libraries were amplified by PCR then purified and size-selected for sequencing with single end 51 bp reads.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
H3K27ac ChIP-seq of WT HCT116 cells with scrambled shRNA replicate 1
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| Data processing |
Basecalls performed using RTA (Illumina)
Reads were aligned to the human genome (hg38) using STAR (v2.2.0c) for ATAC-seq, ChIP-seq, and RNA-Seq. In all cases, only reads that mapped to a unique genomic locations (MAPQ > 10) were used for downstream analysis.
HOMER (v4.8) (http://homer.salk.edu/homer/) was used to process alignment files to generate tag directories and peak files. ATAC-seq peaks were found using the findPeaks program in HOMER with the following options "-style dnase -o auto". ChIP-Seq regions were found using the findPeaks program in HOMER using the paramter "-style histone" to find broad peaks for H3K4me, H3K4me3, H3K27ac, and H3K27me3 using input as background. For RNA-Seq, Fragments Per Kilobase of transcript per milion mapped reads (FPKM) were calculated for annotated genes using HOMER.
Genome_build: hg38
Supplementary_files_format_and_content: peaks.txt file Columns: (1) PeakID (2) chr (3) start (4) end (5) strand (6) Normalized Tag Count (7) Not used (8) findPeaks Score (9) Fold Change vs Local (10) p-value vs Local (11) Clonal Fold Change. regions.txt file columns: (1) PeakID (2) chr (3) start (4) end (5) strand (6) Normalized Tag Count (7) region size (8) findPeaks Score (9) Total Tags (normalized to Control Experiment) (10) Control Tags (11) Fold Change vs Control (12) p-value vs Control (13) Clonal Fold Change. FPKM Tab delimited text file, Columns: (1) Transcript/RepeatID (2) chr (3) start (4) end (5) strand (6) Length (7) Copies (8) Annotation/Divergence (9-16) FPKM for each experiment.
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| Submission date |
Jul 27, 2017 |
| Last update date |
Nov 17, 2022 |
| Contact name |
Diana C. Hargreaves |
| E-mail(s) |
dhargreaves@salk.edu
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| Organization name |
The Salk Institute for Biological Studies
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| Street address |
10010 North Torrey Pines Road
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| City |
San Diego |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (2) |
| GSE101966 |
ARID1A and ARID1B loss in HCT116 cells |
| GSE101975 |
ARID1A and ARID1B loss in HCT116 and TOV21G cells |
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| Relations |
| BioSample |
SAMN07419561 |
| SRA |
SRX3043698 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2719748_Sample25.H3K27ac.ChIP-seq-WT-Rep1.regions.txt.gz |
457.9 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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