|
Status |
Public on Jun 13, 2010 |
Title |
RAG1KO-CTCF-ChIP-Seq |
Sample type |
SRA |
|
|
Source name |
RAG1-/- pro-B cell
|
Organism |
Mus musculus |
Characteristics |
strain: C57BL/6 cell type: pro-B cell chip antibody: CTCF (07-729), DAM1421463 transgenes: none genetic background: RAG1-/-
|
Growth protocol |
Pro-B cells were isolated from femoral bone marrow of Rag1 knockout mice by positive enrichment of B220+ cells using magnetic separation (Miltenyi) and expanded for 10 d in Opti-MEM medium containing 10% FCS, 2x penicillin/streptomycin/glutamine, and 50 µM ß-mercaptoethanol supplemented with IL-7 and SCF.
|
Extracted molecule |
genomic DNA |
Extraction protocol |
Lysates were clarified from sonicated cells or nuclei and protein-DNA complexes were isolated with antibody. Libraries were prepared according to Illumina's instructions. 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 12-18 cycles and library fragments (ChIP) (insert plus adaptor and PCR primer sequences) were size selected from a 2% agarose gel or a 8% polyacrylamide gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer I or Genome Analyzer II following the manufacturer's protocols.
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|
|
Library strategy |
ChIP-Seq |
Library source |
genomic |
Library selection |
ChIP |
Instrument model |
Illumina Genome Analyzer II |
|
|
Description |
Chromatin IP against CTCF in RAG1-/- pro-B cells
|
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.
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|
|
Submission date |
May 24, 2010 |
Last update date |
May 15, 2019 |
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 |
GPL9250 |
Series (1) |
GSE21978 |
A global network of transcription factors, involving E2A, EBF1 and FOXO1, that orchestrates the B cell fate |
|
Relations |
SRA |
SRX021880 |
BioSample |
SAMN00014651 |
Named Annotation |
GSM546526_Sample10.RAG1KO-CTCF.bed.gz |