 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Apr 30, 2019 |
| Title |
Klf4-C2-2iDay3 |
| Sample type |
SRA |
| |
|
| Source name |
Mouse Embryonic Stem Cells
|
| Organism |
Mus musculus |
| Characteristics |
background strain: E14
cell type: Mouse Embryonic Stem Cells
treatment: 2i_Day3
chip antibody: GFP
|
| Growth protocol |
E14Tg2a ESCs were cultured in standard culture medium without feeder and on gelatin coated-dishes. Serum supplemented medium contains Dulbecco's Modified Eagle's Medium (DMEM, Gibco) supplemented with 10% fetal calf serum (FCS, Gibco), L-glutamine (2 nM, Gibco), Na-Pyruvate (1 mM, Gibco), non-essential amino acids (0.1 mM each, Gibco), Penicillin Streptomycin (Gibco), 2-mercaptoethanol (55 µM, Gibco) and LIF (1000 U/ml, Milipore). For serum free culture, cells were maintained in NDiff 227 (StemCells, Inc.) supplemented with MEK inhibitor PD0325901 (1 μM) and GSK3 inhibitor CHIR99021 (3 μM) and LIF (1000 U/ml, Milipore). To test the genome wide binding of Esrrb, Nr5a2 and Klf4, we generated BAC lines expressing the GFP-tagged proteins as described previously by Poser et al (poser I et al Nat. Methods 5, 409–415 (2008). For Nr5a2-null ESCs, cre-recombination was induced by treating with 4-hydroxy tamoxifen (0.5 μM) for 2 days. Cells were confirmed for the correct deletion using PCR. All siRNA experiments were performed for 72h and by using 25pmol stealth siRNAs per well of 6-well plates.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were fixed with 1% formaldehyde (Sigma) in ES-medium. Fixed cells were sonicated using a Diagenode Bioruptor UCD-300 for 4x1 minutes (30 seconds on; 30 seconds off). 50µl of chromatin (500,000 cells) were used with 0.5-1µg of H3K27ac antibody (Diagenode) and incubated overnight at 4ºC with rotation. For TF-ChiP-seq, 15-million cells were used per ChIP together with 15 μg anti-GFP antibody (ab290, Abcam) and 3 ChIP-samples were pooled to yield chromatin equal to 30-million cells per transcription factor. Antibody-chromatin complexes were incubated with protein-A/G magnetic beads, eluted, Supernatant was collected, 8µl 5M NaCl, 3µl proteinase K were added and samples were incubated for 4 hours at 65°C. Finally samples were purified using Qiagen; Qiaquick MinElute PCR purification Kit and eluted in 20µl EB. De-crosslinked DNA was used for library construction using KAPA Hifi kit according to manufacturer’s instruction.
Illumina library preparation was done using the Kapa Hyper Prep Kit. For end repair and A-tailing double stranded DNA was incubated with end repair and A-tailing buffer and enzyme and incubated first for 30 minutes at 20°C and then for 30 minutes at 65°C.Subsequently adapters were ligated by adding 30µl ligation buffer, 10 Kapa l DNA ligase, 5µl diluted adaptor in a total volume of 110µl and incubated for 15 minutes at 15°C.Post-ligation cleanup was performed using Agencourt AMPure XP reagent and products were eluted in 20µl elution buffer. Libraries were amplified by adding 25µl 2x KAPA HiFi Hotstart ReadyMix and 5µl 10x Library Amplification Primer Mix and PCR, 10 cycles. Samples were purified using the QIAquick MinElute PCR purification kit and 300bp fragments selected using E-gel. Correct size selection was confirmed by BioAnalyzer analysis. Sequencing was performed using Illumina HiSeq 2000 machines and generated 43bp single end reads.
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2000 |
| |
|
| Data processing |
ChIP-seq reads were aligned to the mm9 genome assembly using Bowtie2 and reads with mapping quality less than 15 were discarded. PCR duplicates were removed from mapped BAM files and in case of pair-end sequencing, paired fragments were kept only when both reads were mapped uniquely to the same genomic region (unique and concordant alignments). Fragment size modeling was then applied on filtered BAM files (http://code.google.com/p/phantompeakqual-tools/) and peaks were called using MACS2 (http://github.com/taoliu/MACS/) and with default (narrow) settings.
Genome_build: mm9
Supplementary_files_format_and_content: BigWig tracks, data are not normalized for sequencing depth.
|
| |
|
| Submission date |
Dec 14, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Hendrik G Stunnenberg |
| E-mail(s) |
h.stunnenberg@ncmls.ru.nl
|
| Organization name |
Radboud University
|
| Street address |
Geert Grooteplein 30
|
| City |
Nijmegen |
| ZIP/Postal code |
6525GA |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL13112 |
| Series (2) |
| GSE92407 |
Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of pluripotency. (ChIP-seq) |
| GSE92412 |
Epigenetic modulation of a hardwired 3D chromatin landscape in two naive states of pluripotency. |
|
| Relations |
| BioSample |
SAMN06141976 |
| SRA |
SRX2423276 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2429229_YA_Klf4-C2-2iDay3.bw |
173.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
|
|
|
|
 |
