|
| Status |
Public on Dec 31, 2020 |
| Title |
WT_H3K27me3_R2 replicate (ChIP-seq) |
| Sample type |
SRA |
| |
|
| Source name |
WT-HUDEP-2
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HUDEP-2
cell type: erythroblast cells
passage: 20~30
chip antibody: H3K27me3(Cell Signaling Technology, CST#9733)
|
| Growth protocol |
Cells were expanded in StemSpan serum-free expansion medium (SFEM; Stem Cell Technologies) supplemented with 1 µM dexamethasone, 1 µg/mL doxycycline, 50 ng/mL human stem cell factor (SCF), 3 units/mL erythropoietin (EPO), and 1% penicillin–streptomycin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and histone-DNA complexes were isolated with antibody.
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). 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 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 NovaSeq 6000 |
| |
|
| Data processing |
Basecalls performed using CASAVA
BWA (default parameter) was used to align reads to human genome hg19. Duplicated reads were marked by Picard, and uniquely mapped reads were retained by samtools (parameter “-q 1 -F 1024”).
We confirmed quality control results were good following the ENCODE guideline for CHIPSEQ, reads were then extend to the estimated fragment size(by SPP) and converted to bigwig files. We observed clear peaks observed by inspection on Integrated Genomics Viewer (Broad Institute) and there was high consistency among replicates.
Free pipeline to run with DNAnexus available at https://www.stjude.cloud/tools.html. Source code of ChIP-Seq pipeline to deposit on DNAnexus were also available at https://github.com/stjude/ChIP-Seq
Genome_build: hg19
Supplementary_files_format_and_content: bigwig
|
| |
|
| Submission date |
May 30, 2019 |
| Last update date |
Jan 01, 2021 |
| Contact name |
Mitch Weiss |
| E-mail(s) |
mitch.weiss@stjude.org
|
| Phone |
9015953760
|
| Organization name |
st jude children's research hospital
|
| Department |
Hematology
|
| Street address |
262 Danny Thomas Place
|
| City |
Memphis |
| State/province |
Tennessee |
| ZIP/Postal code |
38105 |
| Country |
USA |
| |
|
| Platform ID |
GPL24676 |
| Series (1) |
| GSE115357 |
FBXO11-Mediated Proteolysis of BAHD1 Relieves PRC2-dependent Transcriptional Repression in Erythropoiesis |
|
| Relations |
| BioSample |
SAMN11891724 |
| SRA |
SRX5931553 |
