|
| Status |
Public on May 27, 2010 |
| Title |
BirA-input-GW-ChIP-Seq |
| Sample type |
SRA |
| |
|
| Source name |
BirA RAW264.7 cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: RAW264.7 Macrophage cell line
chip antibody: Streptavidin
treatment: GW3965, 1h
transgenes: BirA
strain: C57BL/6
genotype/variation: wild-type
chip antibody: Streptavidin (NanoLink Streptavidin Magnetic Microspheres (Vendor: SoluLink, cat# M1002-010, lot# LM041609))
|
| Treatment protocol |
GW3965, 1h
|
| Growth protocol |
RAW264.7 Macrophage cell line was cultured in RPMI 1640 containing 10% FCS and 100 U/ml penicillin/streptomycin then treated for 1 hour with 1 µM GW3965.
|
| 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 II |
| |
|
| Description |
Chromatin IP with streptavidin (no BLRP transgene [control]) in RAW264.7 cells treated with GW3965
|
| 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 |
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) |
| GSE21512 |
Simple combinations of lineage-determining transcription factors prime cis-regulatory elements required for macrophage and B cell identities |
|
| Relations |
| SRA |
SRX019779 |
| BioSample |
SAMN00012141 |
| Named Annotation |
GSM538016_Sample36.BirA-input-GW.bed.gz |
