|
| Status |
Public on Jul 07, 2016 |
| Title |
Input chromatin ChIPseq |
| Sample type |
SRA |
| |
|
| Source name |
embryoid bodies
|
| Organism |
Mus musculus |
| Characteristics |
cell type: Runx1-ires-GFP+/Gata1::mCherry+ cells from embryoid bodies after 5 days of differentiation of ES cells towards blood
chip antibody: rabbit IgG Sigma I5006
|
| Growth protocol |
ESCs were grown on gelatinized plates (0.1 % gelatin in water) at 37°C and 5 % CO2 in ESC media (Knockout DMEM (Life Technologies) with 15 % FCS (batch-tested for ESC culture; Life Technologies), 2mM L-glutamine (PAA Laboratories), 0.5 % P/S, 0.1mM β-mercaptoethanol (Life Technologies) and 103 U/ml recombinant LIF (ORF Genetics)). Cells were passaged with TrypLE Express dissociation reagent (Life Technologies) every 1-3 days. Differentiations were performed from single cell suspensions as described by Wilkinson AC et al., 2013 Biology Open. Embryoid bodies were dissociated with TrypLE Express for sorting.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Approximately 7x106 FACS-sorted day 5 EB cells per ChIP were cross-linked using formaldehyde to a final concentration of 1%. As samples were pooled from several sorts, isolated nuclei were frozen on dry ice cold isopropanol and stored at -80°C. During the immunoprecipitation step, 4 μl recombinant histone 2B (New England Biolabs) and 1 μl of mouse RNA (Qiagen; diluted 1/5 in IP dilution buffer) were added as carriers, followed by 7 μg of primary antibody
Sequencing libraries were prepared using the TruSeq Kit (Illumina) for high throughput sequencing on an Illumina HiSeq 2500, according to manufacturer’s instructions, with size selection for fragments of 150-400 bp.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Low cell number for this ChIP sample meant that there was insufficient material to do an IgG ChIP.
|
| Data processing |
ChIP-seq reads aligned to mm10 using bowtie2 (version 2.0.6) with extra parameters (-k 2 -N 1).
bigWig density profiles were created with in-house script incorporating UCSC's wigToBigWig utility.
Peaks were called using macs2 (version 2.0.10).
Genome_build: mm10
Supplementary_files_format_and_content: BigWig – read density profile, BED – peaks file
|
| |
|
| Submission date |
Nov 13, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Evangelia Diamanti |
| E-mail(s) |
ed347@cam.ac.uk
|
| Phone |
01223 62317
|
| Organization name |
University of Cambridge
|
| Department |
Haematology
|
| Lab |
Gottgens
|
| Street address |
Wellcome Trust / MRC Building, Hills Road
|
| City |
Cambridge |
| ZIP/Postal code |
CB2 0XY |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE74994 |
Single Cell Expression Profiling Resolves the Transcriptional Programs of Early Mesoderm Diversification |
|
| Relations |
| BioSample |
SAMN04267192 |
| SRA |
SRX1431660 |
