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Sample GSM1666208 Query DataSets for GSM1666208
Status Public on Jul 21, 2015
Title GM12878_K27ac_direct_100_Rep1
Sample type SRA
Source name GM12878
Organism Homo sapiens
Characteristics cell type: B-lymphocyte
chip antibody: H3K27ac (ab4729, Abcam, Cambridge, MA, USA)
Treatment protocol None
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.
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.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2500
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
Submission date Apr 24, 2015
Last update date May 15, 2019
Contact name Kai Tan
Organization name The University of Iowa
Department Internal Medicine
Street address 3292 CBRB, 285 Newton Road
City Iowa City
State/province IA
ZIP/Postal code 52242
Country USA
Platform ID GPL16791
Series (1)
GSE65516 A microfluidic device for epigenomic profiling using 100 cells
BioSample SAMN03567108
SRA SRX1008314

Supplementary file Size Download File type/resource 99.0 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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