|
| Status |
Public on Apr 17, 2017 |
| Title |
H1_2 |
| Sample type |
SRA |
| |
|
| Source name |
H1 POU5F1-IRES-eGFP
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: H1 POU5F2-eGFP reporter cells
|
| Treatment protocol |
Regular hESC culture without any special treatment.
The core promoter of MSH5 and PRRC2A was deleted by a pair of CRISPR/Cas9 targeting- 150bp to +100bp region near TSS. After picking single clones and Sanger sequencing verification of the genotype, the mutant cells and wild-type control cells were subjected to ATAC-seq analysis.
|
| Growth protocol |
Cells were cultured at 37 celsius degree in 5% CO2, in Essential 8 media (Stemcell Technologies).
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
ATAC-seq was performed by following the protocol described earlier (Buenrostro et al, Nature Method, 2013).
Briefly, each library starts with 100k cells which were permeabilized with NPB (0.2% NP-40, 5%BSA, 1Mm DTT in PBS with one complete proteinase inhibitor) at 4 degree for 10min, followed by spin down at 500g for 5min. The resulting nuclei were resuspended in 20ul 1xDMF (33mM Tris-acetate (pH=7.8), 166mM K-Acetate, 10mM Mg-Acetate, 16 % DMF). The chromatin tagmentation was done by adding 0.5ul Tn5 into 10ul solution for 30min at 37 degrees, followed by standar Illumina Tru-seq library prep protocol.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Description |
ATAC-seq using WT and mutant POU5F1-eGFP reporter hESC.
POU5F1-eGFP H1 hESC reporter cells purchased from WiWell Institute.
|
| Data processing |
Library strategy: ATAC-seq
We processed our ATAC-seq data in the following steps: 1) ATAC-seq sequencing reads were mapped to hg19 reference genome using Bowtie(61) in pair-end mode; 2) poorly mapped, improperly paired and mitochondrial reads were filtered; 3) PCR duplications were further removed using Picards MarkDuplicates (http://broadinstitute.github.io/picard.); 4) Mapping positions of reads were adjusted accounting for Tn5 insertion; 6) Reads were next shifted for 75bp followed by peak calling using MACS2(71) with following parameters “-q 0.01 --nomodel --shift 175 –B --SPMR --keep-dup all --call-summits”; 7) ATAC-seq signal was normalized into RPKM using deeptools(72) for visualization.
Genome_build: hg19
Supplementary_files_format_and_content: .bw
|
| |
|
| Submission date |
Nov 09, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Bing Ren |
| Organization name |
University of California, San Diego School of Medicine
|
| Street address |
9500 Gilman Dr., CMM-East, Admin Area/Rm 2071
|
| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92093 |
| Country |
USA |
| |
|
| Platform ID |
GPL20301 |
| Series (1) |
| GSE81026 |
Cis Regulatory Element Scan by Tiling-deletion and Sequencing |
|
| Relations |
| BioSample |
SAMN06006844 |
| SRA |
SRX2338897 |
