|
| Status |
Public on Feb 22, 2016 |
| Title |
H3K27ac ChIP-seq |
| Sample type |
SRA |
| |
|
| Source name |
iPSC-derived cardiomyocytes
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: iPSC-derived cardiomyocytes
passages: 20-30
iPSc source: individual ID: YRI-NA18852
antibody: Abcam – cat# Ab4729
|
| Growth protocol |
iPSC-derived cardiomyocytes were generated as in Burridge et al (2014): Lymphoblastoid cell line (LCL)-derived iPSCs (passage 20-30) were grown in Essential 8 media (Invitrogen) to 80-90% confluency and then treated with a GSK-3 inhibitor (CHIR99021, Tocris) for 24 hours. 48 hours later, the cells were treated with a Wnt antagonist (Wnt-C59, Tocris) for 48 hours and then maintained in general heart media (RPMI supplemented with B27-minus Insulin, Invitrogen). On day 15 of the differentiation, cardiomyocytes were selected using media supplemented with lactate and then 4 days later the cells were harvested. Aliquots of 5 million cells were immediately crosslinked in 1% formaldehyde for 10 minutes following the protocol provided by Rao et al. (2014). An additional 1-2 million cells were used in flow cytometry to assess purity using both TNNT2 (Troponin T) and TNNI3 (Troponin I) antibodies. Cells used in Hi-C were > 80% positive for both markers.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Histone-DNA or PolII-DNA complexes were isolated with antibody.
Libraries were prepared according to Illumina's instructions provided with the TrueSeq ChIP Sample Preparation Kit (Catalog #: IP-202-1012). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. Coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250-450 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
H3K27ac ChIP-seq
|
| Data processing |
Basecalls performed with Illumina bcl2fastq2-v2.17.1.14 by the Functional Genomics Facility at the University of Chicago
Reads were aligned to hg19 with bowtie2-2.2.3
Reads with MAPQ < 10 were filtered out
HOMER was used to call peaks using FDR < 0.001 and the region and histone settings. H3K27me3 peaks were called with FDR < 0.005 and -size 500
Genome_build: hg19
Supplementary_files_format_and_content: bed files were parsed from HOMER's output and contain peaks called
|
| |
|
| Submission date |
Jan 27, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Noboru Jo Sakabe |
| Organization name |
University of Chicago
|
| Department |
Human Genetics
|
| Street address |
920 E 58th st
|
| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60615 |
| Country |
USA |
| |
|
| Platform ID |
GPL20301 |
| Series (1) |
| GSE77267 |
Genome-wide map of 4 histone modifications and PolII in iPSC-derived human cardiomyocytes |
|
| Relations |
| BioSample |
SAMN04442277 |
| SRA |
SRX1548293 |
