|
| Status |
Public on May 27, 2010 |
| Title |
E2AKO-PU.1-E2A-ER-ChIP-Seq |
| Sample type |
SRA |
| |
|
| Source name |
E2A-/- pre-pro-B cell
|
| Organism |
Mus musculus |
| Characteristics |
cell type: pre-pro-B cell
chip antibody: PU.1 (sc-352)
treatment: The cells were spin-infected with a retrovirus expressing full length E2A fused to the estrogen receptor ligand binding domain, as well as hCD25 from a second cistron. After 16 h in culture, cells were treated with tamoxifen (1 µM final concentration) for 6 h. Cells expressing hCD25 were then enriched magnetically (Miltenyi).
transgenes: E2A-ER
strain: C57BL/6
genotype/variation: E2A-/-
chip antibody: PU.1 (Vendor: Santa Cruz, cat# sc-352, lot# F1808)
|
| Treatment protocol |
The cells were spin-infected with a retrovirus expressing full length E2A fused to the estrogen receptor ligand binding domain, as well as hCD25 from a second cistron. After 16 h in culture, cells were treated with tamoxifen (1 µM final concentration) for 6 h. Cells expressing hCD25 were then enriched magnetically (Miltenyi).
|
| Growth protocol |
E2A-/-and EBF-/- pre-pro-B cells were cultured on S17 stromal cells in the presence of IL7 (10 ng/ml), SCF (10 ng/ml), and FLT3 ligand (10 ng/ml) in Iscove’s Dulbecco’s medium and in the presence of 10% FCS, 200 U/ml penicillin, 200 mg/ml streptomycin and 4 mM L-glutamine.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Lysates were clarified from sonicated nuclei and protein-DNA complexes were isolated with antibody. Alternatively, DNA was phenol/chloroform-extracted from MNase-treated nuclei, and 134-154 bp long fragments were extractred from a 2% agarose gel. 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, ChIP DNA was PCR-amplified with Illumina primers for 12-16 cycles and library fragments of 200-300 bp (ChIP) (insert plus adaptor and PCR primer sequences) were isolated from a 2% agarose gel. For MNase-Seq, size-selected mononucleosome-derived DNA fragments were ligated to modified adapters that incorporate both the adapter sequence as well as the primer landing sites for the flow cell-conjugated primers. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer or Genome Analyzer II following the manufacturer's protocols.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer |
| |
|
| Description |
Chromatin IP against PU.1 in E2A-/- pre-pro-B cells expressing an Tamoxifen activated E2A (E2A-ER)
|
| Data processing |
Alignment: Reads were truncated to 25 bp and aligned to the mouse mm8 genome (NCBI Build 36) using eland. Only reads that mapped to a single, unique position were used for downstream analysis.
Peak Identification: Peaks were identified using custom software (HOMER, available at http://biowhat.ucsd.edu/homer/). For transcription factors, peaks were found by identifying 200 bp regions with tag density exceeding a threshold set at a 0.1% false discovery rate (determined by tag randomization). Only peaks with >4x normalized tag density relative to control sequencing and >4x normlaized tag density to the sourrounding 10kb of region were considered for analysis.
|
| |
|
| Submission date |
Apr 27, 2010 |
| Last update date |
Jan 08, 2015 |
| Contact name |
Christopher Benner |
| E-mail(s) |
cbenner@ucsd.edu
|
| Organization name |
University of California, San Diego (UCSD)
|
| Department |
Medicine
|
| Street address |
9500 Gilman Dr. MC 0640
|
| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92093-0640 |
| Country |
USA |
| |
|
| Platform ID |
GPL9185 |
| Series (1) |
| GSE21512 |
Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities |
|
| Relations |
| BioSample |
SAMN02196114 |
| Named Annotation |
GSM537998_Sample16.E2AKO-PU.1-E2A.bed.gz |
