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Sample GSM2417329 Query DataSets for GSM2417329
Status Public on Jun 13, 2018
Title Supt16_ChIP_seq_rep1,rep2
Sample type SRA
Source name mESCs
Organism Mus musculus
Characteristics cell line: E14 cell line
Growth protocol The E14 cell line (mESCs) was cultured at 37 °C, 7.5% CO2, on 0.1% gelatin coated plates, in DMEM + GlutaMax™ (Gibco) with 15% fetal bovine serum (Gibco), MEM non- essential amino acids (Gibco), penicillin/streptomycin (Gibco), 550 µM 2-mercaptoethanol (Gibco), and 10 ng/ml of leukaemia inhibitory factor (LIF) (eBioscience).
Extracted molecule genomic DNA
Extraction protocol Cells were crosslinked with 1% formaldehyde for 20 min followed by quenching for 5 min with the addition of glycine to a final concentration of 0.125 M. After washing with PBS buffer, cells were collected and lysed in Cell Lysis buffer (5 mM Tris pH8.0, 85 mM KCl, 0.5% NP40 ) with proteinase inhibitors (10 µl/mL Phenylmethylsulfonyl fluoride (PMSF), 1 µl/mL Leupeptin and 1 µl/mL Pepstatin). Pellets were spun for 5 min at 5000 rpm at 4°C. Nuclei were lysed in Nuclei Lysis Buffer (1% SDS, 10 mM EDTA, 50 mM Tris HCl) and samples were sonicated for 12 min. Samples were centrifuged for 20 min at 13,000 rpm at 4°C and the supernatant was diluted in IP buffer (0.01% SDS, 1.1% Triton-X-100, 1.2 mM EDTA, 16.7 mM Tris HCl, 167 mM NaCl) and the appropriate antibody was added and left overnight with rotation at 4°C. Protein A/G Dynabeads (Invitrogen) were added for 1h and after extensive washed samples were eluted in Elution Buffer (1% SDS, 0.1 M NaHCO3). 20 µL of 5 M NaCl were added and samples were reverse-crosslinked at 65°C for 4h. Following phenol-chloroform extraction and ethanol precipitation, DNA was incubated at 37°C for 4h with RNAse (Sigma).
Approximately 10-20 ng of ChIP material was used for library preparation. End-repair and adaptor ligation was prepared as described previously with a few modifications (Tessarz, et al). Double sided size selections (~200 – 650bp) were performed using the MagSI-NGS Dynabeads (MagnaMedics, #MD61021) according to the manufacturer’s instructions. Purified adapter-ligated ChIP material was run on a high sensitivity DNA chip on a 2200 TapeStation (Agilent) to assess size distribution and adaptor contamination.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2500
Description ChIP antibody: Anti-Supt16 (Cell Signaling)
Data processing ChIP-seq files were aligned using Bowtie2 (v 2.2.6). BAM files were converted to bigwig (10 bp bin) and normalised to x1 sequencing depth. Duplicated reads were removed.
Genome_build: mm10
Submission date Dec 05, 2016
Last update date May 15, 2019
Contact name Peter Tessarz
Organization name Max Planck Institute
Street address Joseph-Stelzman-Str 9b
City Cologne
State/province Nordrhein-Westfalen
ZIP/Postal code 50931
Country Germany
Platform ID GPL17021
Series (1)
GSE90906 Transcriptional repression by FACT is linked to regulation of chromatin accessibility at the promoter of ES cells
BioSample SAMN06111831
SRA SRX2396607

Supplementary file Size Download File type/resource 344.5 Mb (ftp)(http) BW 116.5 Kb (ftp)(http) TAB
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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