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| Status |
Public on Mar 01, 2015 |
| Title |
GM19201 H3K27ac |
| Sample type |
SRA |
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| Source name |
Lymphoblastoid cell lines
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| Organism |
Homo sapiens |
| Characteristics |
cell line: GM19201
antibody: H3K27ac
antibody manufacturer: Abcam
antibody catalog number: AB4729
antibody lot number: 730178
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| Biomaterial provider |
Coriell Cell Repositories http://ccr.coriell.org/Sections/Search/Search.aspx?PgId=165&q=GM19201
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| Treatment protocol |
Approximately ten million cells from each cell line were cross-linked with 1% formaldehyde for 15 minutes.
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| Growth protocol |
Cell lines were purchased from Coriell Institute for Medical Research and grown and maintained according to the provided Coriell protocols. Briefly, cells were grown in RPMI 1640 media (15% FBS, 2mM L-glutamine) to approximately 1 million cells per ml and then collected for ChIP-seq or snap-frozen in liquid nitrogen and stored at -80C.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Excess formaldehyde was quenched by addition of glycine (0.625M). Cells were washed with cold PBS and spun down (3,000 rpm for 10 minutes at 4°C; Kubota 3500). The pellet was re-suspended in FA lysis buffer (0.25% Triton X-100, 10 mM EDTA, 10 mM Tris HCl [pH 8.0], 100 mM NaCl, Roche 1X cOmplete protease inhibitor) and incubated for 15 minutes. Nuclei were collected (3,000 rpm for 10 minutes at 4°C; Kubota 3500) and re-suspended in 300 μl SDS lysis buffer (1% SDS, 1% Triton X 100, 2 mM EDTA, 50 mM Hepes-KOH [pH 7.5], 0.1% Na dodecyl sulfate, Roche 1X cOmplete protease inhibitor). Nuclei were lysed for 15 minutes, after which sonication was used to fragment chromatin to an average size of 200–500 bp (Bioruptor Next gen, Diagenode). Cellular debris was removed by centrifugation at 15,000 rpm at 4°C (Kubota 3500). Next, 300 μl of nuclear lysate was diluted 1:10 with a dilution buffer (1% Triton X 100, 2 mM EDTA, 50 mM Hepes-KOH [pH 7.5], 0.1% Na dodecyl sulfate, Roche 1X cOmplete protease inhibitor) and protein-DNA complexes were immuno-precipitated using 3 μg of H3K27acetyl antibody (AB4729, Lot number 730178, Abcam) coupled to 50μl protein G Dynal beads (Invitrogen) overnight. The beads were washed and protein-DNA complexes were eluted with 150 μl of elution buffer (1% SDS, 10 mM EDTA, 50 mM Tris.HCl [pH 8]), followed by protease treatment and de-crosslinking at 65°C overnight. After phenol/chloroform extraction, DNA was purified by ethanol precipitation.
80% of ChIP material was used for library preparation using the Biooscientific library preparation kit with the following modification. The provided adapters were diluted down 40-fold in order to prevent adapter dimer formation that would affect enrichment. Libraries were then enriched for 15 cycles with PFX polymerase (Invitrogen) followed by gel size selection (300-500bp). The ChIP libraries were then sequenced on the Illumina Hiseq 2000 platform in three batches
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
technique: RegVAR-seq
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| Data processing |
Illumina Casava1.7 software was used for basecalling 2x100 and 1x100 libraries; Illumina Casava1.8 software was used for basecalling 2x50 libraries
ChIP-seq reads were aligned to hg19 reference genome with bwa 0.6.2.r126 (-q 10 option)
Peaks were called using Dfilter (Kumar et al., Nature Biotech 2013)
Genome_build: hg19
Supplementary_files_format_and_content: wig files of uniquely mapped reads were created using custom perl scripts and converted to bigWig using wigToBigWig
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| Submission date |
Jun 26, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Ricardo Cruz-Herrera del Rosario |
| E-mail(s) |
delrosariorc@gis.a-star.edu.sg
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| Phone |
+65-6808-8240
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| Organization name |
Genome Institute of Singapore
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| Department |
Computational and Systems Biology
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| Street address |
60 Bipolis St, #02-01 Genome
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| City |
Singapore |
| ZIP/Postal code |
138672 |
| Country |
Singapore |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE58852 |
Sensitive detection of chromatin-altering polymorphisms reveals autoimmune disease mechanisms |
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| Relations |
| BioSample |
SAMN02887220 |
| SRA |
SRX627261 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1420887_GM19201_GIS_prabhakar_2x50.bw |
57.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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