|
| Status |
Public on May 26, 2023 |
| Title |
ERG_P199L_H3K27me3 |
| Sample type |
SRA |
| |
|
| Source name |
Hoxb8-ER cells
|
| Organism |
Mus musculus |
| Characteristics |
transfection: ERG_P199L
strain: C57BL/6
cell type: myeloid progenitors
chip antibody: H3K27me3 (Abcam)
|
| Growth protocol |
ChIP material was prepared from ER-Hoxb8 cells stably expressing ERG variants and an empty vector for control. 10^7 cells were used per sample
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were crosslinked with 1% paraformaldehyde for 15 minutes and were quenched with glycine for 5 minutes at room temperature. Lysis buffer with protease inhibitor was gently added to cell pellet. Fixed chromatin was sonicated with the Bioruptor Pico using pre-optimised conditions and immunoprecipitated with the indicated antibody. Immunoprecipitation was preform using polyclonal antibody raised against H3K27ac, H3K4me1, H3K4me3, H3K9ac (Abcam). As a control, nonspecific rabbit IgG (I5006; Sigma Aldrich) was used.
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 Illumina HiSeq 2500 following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Data processing |
Adapters were trimmed using the cutadapt tool. Following adapter removal, reads that were shorter than 30 nucleotides were discarded (cutadapt option –m 30).
The reads were aligned uniquely to the mouse genome (mm10) using bowtie.
Bound regions were detected using MACS2.
Bedtool commands were used for merging and intersections between peak files.
Assembly: mm10
Supplementary files format and content: Merged peaks for each Histon mark from all conditions in excel file
Supplementary files format and content: bigwig files were generated using MACS2
|
| |
|
| Submission date |
Apr 07, 2022 |
| Last update date |
May 26, 2023 |
| Contact name |
Eitan Kugler |
| E-mail(s) |
et.kugler@gmail.com
|
| Organization name |
Rabin medical center
|
| Department |
Hematology
|
| Street address |
Zeev Jabutinsky Rd 39
|
| City |
Petah Tikva |
| State/province |
Please make your selection |
| ZIP/Postal code |
49100 |
| Country |
Israel |
| |
|
| Platform ID |
GPL17021 |
| Series (2) |
| GSE200389 |
The NCOR-HDAC3 co-repressive complex modulates the leukemogenic potential of ERG. |
| GSE200393 |
A single amino acid at the PNT domain of ERG mediates its leukemogenic activity through interaction with the NCoR-HDAC3 co-repressor complex. |
|
| Relations |
| BioSample |
SAMN27403363 |
| SRA |
SRX14775877 |
