|
| Status |
Public on Aug 17, 2012 |
| Title |
Input_async_ChIPseq |
| Sample type |
SRA |
| |
|
| Source name |
asynchrnous G1E-ER4+E2 cells
|
| Organism |
Mus musculus |
| Characteristics |
strain: 129
developmental stage: Rescued subline of G1E expressesing a Gata1-estrogen receptor (ER) transgene; recapitulates erythroid differentiation after induction by estradiol treatment for 24hr.
chip antibody: None
|
| Treatment protocol |
For asynchronous ChIP, G1E-ER4 cells were induced with 100 nM estradiol for 24 hours. Cells were crosslinked with 1% (0.33 M) formaldehyde in PBS at room temperature for 10 min and quenched with 0.125 M glycine for 5 min
For mitotic ChIP, G1E-ER4 cells were induced with 100 nM estradiol for 18 hours. Nocodazole was added (200 ng/mL) for 6-7 hours before harvest. Cells were crosslinked with 1% (0.33 M) formaldehyde in PBS at room temperature for 10 min and quenched with 1 M glycine for 5 min .Cells were stained with anti-H3S10ph antibodies and Dy649-conjugated anti-rabbit IgG F(ab’)2 antibody fragments in PBS/2% FBS/2 mM EDTA, 1 mM PMSF, and protease inhibitor cocktail (Sigma). 107 H3S10ph-positive cells per IP were sorted on a FACSAria II (Beckton Dickinson).
|
| Growth protocol |
G1E-ER4 cells were grown in IMDM media with 15% fetal calf serum 2U/ml erythropoietin (Amgen’s EpoGen) and 50ng/ml stem cell factor.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
For ChIP-seq, ChIP DNA or input DNA was amplified by Illumina ChIP-Seq library preparation kit. To prepare the sequencing libraries, the ChIP DNA fragments were repaired to generate blunt ends, with a single A nucleotide adding to each end. Double-stranded Illumina adaptors were ligated to both ends of the fragments. Ligation products were amplified by 18 cycles of PCR, and the PCR products between 250 and 300 bp were gel purified. Libraries were quantified with a Quant-iT dsDNA HS Assay Kit.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
Rescued subline of G1E expressing a Gata1-estrogen receptor (ER) transgene; recapitulates erythroid differentiation after induction by estradiol treatment for 24hr.
Chomatin IP input, GSM453998
|
| Data processing |
Reads generated from a sample ran on more than one lane were concatenated
Concatenated ChIP-seq reads were aligned to the mm9 genome assembly using bowtie version 0.12.7 with default settings on the Galaxy platform
Artifactual reads mapping to the coding exons of GATA1, BRD3,GATA2,ZFPM1 were filtered using Samtools.
Peaks were called on filtered mapped reads with repsective inputs as ChIP-seq control file using MACS 1.3 with the option mfold 10 on the Galaxy platform
Genome_build: mm9
Supplementary_files_format_and_content: Alignment files contain reads mapped to mm9 build for each sample. Peak files are bed format files containing MACS identified GATA1 occupied regions for the indicated samples.
|
| |
|
| Submission date |
Mar 17, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Ross Hardison |
| E-mail(s) |
rch8@psu.edu
|
| Organization name |
Pennsylvania State University
|
| Street address |
303 Wartik Lab
|
| City |
University Park |
| State/province |
PA |
| ZIP/Postal code |
16802 |
| Country |
USA |
| |
|
| Platform ID |
GPL11002 |
| Series (1) |
| GSE36589 |
Genome-wide map of transcription factor GATA1 occupancy during mitosis in G1E ER4+E2 cells |
|
| Relations |
| Reanalysis of |
GSM453998 |
| Reanalyzed by |
GSM3665841 |
| Reanalyzed by |
GSM3665995 |
| SRA |
SRX130175 |
| BioSample |
SAMN00829002 |
