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Sample GSM2417330

Query DataSets for GSM2417330

Status

Public on Jun 13, 2018

Title

Input_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. 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

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

E-mail(s)

ptessarz@age.mpg.de

Organization name

Max Planck Institute

Street address

Joseph-Stelzman-Str 9b