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| Status |
Public on Jul 21, 2015 |
| Title |
GM12878_K4me3_direct_600_Rep2 |
| Sample type |
SRA |
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| Source name |
GM12878
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| Organism |
Homo sapiens |
| Characteristics |
cell type: B-lymphocyte
chip antibody: H3K4me3 (07-473, Millipore,Billerica, MA, USA)
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| Treatment protocol |
None
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| Growth protocol |
GM12878 cells were grown in RPMI 1640 medium (Gibco) supplemented with 15% Fetal Bovine Serum (FBS) (Gibco), penicillin (Invitrogen), streptomycin (Invitrogen). Cultures were seeded at a concentration of between 200,000 and 500,000 viable cells per mL. Medium was changed every 2-3 days depending on cell density.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
The procedure was different for preparing sonicated chromatin from 100 or 600 cells directly. Cells were counted with a hematocytometer and 100/600 cells were transferred to a 1.5 ml LoBind Eppendorf tube containing 10 µl 10% FBS in PBS. Cells were then cross-linked for 5 min at room temperature by adding 0.625 µl 16% formaldehyde to yield a final concentration of 1%. Cross-linking was quenched by adding 1.25 µl 2.5 M glycine for 5 min at room temperature. The cross-linked sample was then diluted using 120 µl Covaris sonication buffer (to give a total volume of 130 µl) and sonicated with a Covaris E220 sonicator for 8 min with 5% duty, 105 peak incident power and 200 cycles per burst in a Covaris microtube (520045, Covaris). The sonicated lysate was centrifuged at 14000×g for 10 min at 4°C. Sonicated chromatin in the supernatant was transferred to a new 1.5 ml LoBind Eppendorf tube for MOWChIP-Seq.
The ChIPed and input DNA was constructed into libraries by using the ThruPLEX FD Kit (Rubicon Genomics, Ann Arbor, MI, USA). During the amplification step, 11cycles was needed to get enough library from input DNA without over-amplification. 12~13 cycles is needed for ChIPed DNA extracted from 10000 cells. 14~15 cycles is normally required for ChIPed DNA from 1000 or less cells.
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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 sequencing reads were mapped to the human genome (hg19) or mouse genome (mm9) using Bowtie2 (v2.2.2) with default parameter setting. Only uniquly mapped reads were used for analysis
ChIP-seq peaks were identified by two peak caller, MACS (p-value < 10-5) and SPP (z-score > 4) with default parameter setting.
To generate the wig files, the human or mouse genome was divided into 100 bp bins, and the number of uniquely mapped reads were counted in each bin. We normalized the tag counts in each bin according to the total number of uniquely mapped reads. Input reads were processed in the same way and their normalized signals were subtracted from the ChIP-seq tracks.
Normalized wig files were converted to binary bigwig files
Genome_build: hg19 and mm9
Supplementary_files_format_and_content: bigwig files
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| Submission date |
Apr 24, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Kai Tan |
| Organization name |
The University of Iowa
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| Department |
Internal Medicine
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| Street address |
3292 CBRB, 285 Newton Road
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| City |
Iowa City |
| State/province |
IA |
| ZIP/Postal code |
52242 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE65516 |
A microfluidic device for epigenomic profiling using 100 cells |
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| Relations |
| BioSample |
SAMN03567103 |
| SRA |
SRX1008309 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1666203_GM12878_K4me3_Undiluted_600_Rep2.bw |
99.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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