 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jan 30, 2017 |
| Title |
DE_Input rep1 |
| Sample type |
SRA |
| |
|
| Source name |
Definitive Endoderm
|
| Organism |
Mus musculus |
| Characteristics |
cell type: ES_derived Definitive Endoderm
strain: 129/Sv//EV
day of differentiation: Day 5
chip antibody: Nil
|
| Treatment protocol |
ES cells (ESC) were seeded in suspension at low density (1x104 cells/ml) in the absence of LIF to form embryoid bodies (EBs). ES cells were seeded in DMEM (Invitrogen) supplemented with 15% fetal calf serum (Gibco), 1% Penicillin/Streptomycin (Invitrogen), 0.1mM 2-mercaptoethanol (Sigma), 1% Glutamine (Invitrogen), 1% MEM Non-essential amino acids (Gibco) and 1mM Sodium Pyruvate (Sigma). On day 2 EBs were transferred into N2B27 medium (Cellartis) supplemented with 20ng/ml ActivinA (R&D systems) and 20ng/ml EGF (Peprotech) to induce DE differentiation (Morrison et al. 2008).
|
| Growth protocol |
Definitive Endoderm differentiation from ESCs cultured as EBs for 2 days, induced to differentiate to a DE fate in N2B27 medium supplemented with ActivinA and EGF. EBs were dissociated by incubation with 0.25% trypsin (Gibco) for 3 min at 37˚C with constant agitation followed by gentle pipetting to obtain a single cell suspension.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Fixed cells were lyzed on ice for 20 minutes (5 mM PIPES, 85 mM KCl, 0.5% Igepal-CA 630) before pelleting and nuclear lysis (50 mM Tris-HCl, 10 mM EDTA, 1% SDS), chromatin was then sonicated to 200bp in a Covaris S220 and incubated overnight with 2 µl of anti-H3K4me3 (07-473; Millipore) and Protein A and Protein G Dynabeads (Invitrogen). Beads were washed seven times with RIPA buffer variants (10 mM Tris-HCl, 1mM EDTA, 0.5mM EGTA, 1% Triton X-100, 0.1% SDS, 0.1% Sodium Deoxycholate): RIPA (twice), High Salt RIPA (500 mM NaCl; twice), RIPA with 250mM LiCl (once) and T.E. Buffer (twice) before treatment with RNAse A at 37ºC for 1 hr (Roche) and Proteinase K 65ºC overnight (Thermo Fisher). DNA was recovered by phenol-chloroform extraction.
Libraries were indexed for sequencing using NebNext Ultra II (New England BioLabs); library profiles were visualized using D1000 tape on a TapeStation (Agilent Technologies) and quantified using a universal library quantification kit (KAPA Biosystems).
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Mapping : bowtie -m 2 --maxIns 350
For un-mapping reads : trimming adaptors trim_galore -- length 10, combining overlapping R1-R2 reads : flash -m 9 -x 0.125
Combining trimmed+flashed and originally mapped reads, mapping again with bowtie -m 2 --maxIns 350
Removing read pairs overlapping Blacklisted regions
Removing duplicates : samtools rmdup
Reconstructing sequenced fragments from R1+R2 read pairs ( bedtools bedpe | cut -f 1,2,6) - for visualisation
Pileup of fragments (bedtools genomecov -counts)
Generating bigwig tracks (ucsctools bedGraphToBigWig)
Processing: Plot of density of alignments in a moving 300 bp window with an increment of 30 bp
Genome_build: mm9
Supplementary_files_format_and_content: bigWig : un-normalised coverage of filtered fragments (reconstructed, duplicate-filtered fragments : see above), density of alignments in a moving 300 bp window with an increment of 30 bp
|
| |
|
| Submission date |
Jan 25, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Damien Downes |
| E-mail(s) |
[email protected]
|
| Phone |
01865222374
|
| Organization name |
The University of Oxford
|
| Department |
MRC Weatherall Institute of Medicine
|
| Street address |
John Radcliffe Hospital
|
| City |
Headington |
| State/province |
Oxfordshire |
| ZIP/Postal code |
OX3 9DU |
| Country |
United Kingdom |
| |
|
| Platform ID |
GPL19057 |
| Series (2) |
| GSE94048 |
Eomes related gene regulation in Definitive Endoderm (ChIP-seq) |
| GSE94250 |
Eomes related gene regulation in Definitive Endoderm |
|
| Relations |
| BioSample |
SAMN06279428 |
| SRA |
SRX2527268 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2467512_DE_Input_rep1.bw |
266.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
