Sample information curated by ChIP-Atlas

Antigen

Antigen Class
Input control
Antigen
Input control

Cell type

Cell type Class
Muscle
Cell type
C2C12
Primary Tissue
Skeletal Muscle
Tissue Diagnosis
NOS

Attributes by original data submitter

Sample

source_name
C2C12
antibody
none
cell line
C2C12
treatment
none

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
FA (Sigma) was added to culture medium to a final concentration of 1%. Crosslinking was allowed to proceed for 10 min at RT and stopped by addition of glycine at a final concentration of 0.125 M. Fixed cells were washed and harvested with PBS. Cells were lysed in Buffer 1 (50 mM Hepes/KOH pH 7.5; 140 mM NaCl; 1 mM EDTA; 10% Glycerol; 0.5% NP-40; 0.25% Triton). The resulting pellet was resuspended in Buffer 2 (200 mM NaCl; 1mM EDTA; 0.5mM EGTA; 10 mM Tris pH 8). Each resuspension step was followed by 10 min incubation at 4°C. Nuclei were then pelleted by centrifugation, resuspended in Buffer 3 (50 mM Tris pH 8; 0.1% SDS; 1% NP-40; 0.1% Na-Deoxycholate; 10 mM EDTA; 150 mM NaCl). All buffers were supplemented with phosphatase and protease inhibitors (Sigma) prior to usage. Samples were sonicated with Bioruptor Power-up (Diagenode) yielding genomic DNA fragments with a bulk size of 150-300 bp. Chromatin was precleared with Protein A/G ultralink beads (#53133, Thermo Fisher Scientific) for 1h at 4°C and immunoprecipitation with the specific antibodies overnight at 4°C. Immunocomplexes were recovered by adding protein A/G ultralink beads, preblocked with BSA and incubated for 2 h at RT. Beads were washed twice with Low salt buffer (0.1% SDS; 1% Triton; 2 mM EDTA; 20 mM Tris pH 8; 150 mM NaCl), twice with High salt buffer (0.1% SDS; 1% Triton; 2 mM EDTA; 20 mM Tris pH 8; 500 mM NaCl), once with LiCl buffer (10 mM Tris pH 8.0; 1% Na-deoxycholate; 1% NP- 40, 250 mM LiCl; 1 mM EDTA) and twice with TE buffer + 50mM NaCl. All buffers were supplemented with phosphatase and protease inhibitors (Sigma). Chromatin was eluted with Elution buffer (50mM Tris, pH8; 1mM EDTA; 1%SDS; 0,2M NaCl) at 65°C and crosslinking was reversed O/N at 65°C using Proteinase K (Sigma). The eluted material was phenol/chloroform-extracted and ethanol-precipitated Five to fifteen ng of ChIPed DNA or whole cell extract (Input) were prepared for sequencing on Illumina Hiseq 2000. We used the library kit (Truseq DNA sample prep kit V2, Illumina) according to the manufacturers' instructions with the following modifications: DNA fragments were repaired to blunt ends, purified with magnetic beads (Agencourt AMPure XP, Beckman coulter) and a step of A-tailing was performed before ligating to Illumina adapters. Two steps of DNA purification on magnetic beads were carried out to eliminate non-ligated adaptors and then amplified with 15 PCR cycles. To remove non-ligated left adapters and large DNA fragments, DNA libraries were selected on E-Gel (2% SizeSelect, Invitrogen) to obtain 280-330 bp DNA fragments (including 130 bp of adapters). To ensure high quality of the library, samples were tested using the DNA high sensitivity chip (Agilent Technologies) and for positive target enrichment by qPCR. Furthermore, samples were then quantified by qPCR and PicoGreen (Qubit® 2.0 Fluorometer, Invitrogen)

Sequencing Platform

instrument_model
Illumina HiSeq 2000

mm10

Number of total reads
48453695
Reads aligned (%)
96.7
Duplicates removed (%)
22.1
Number of peaks
528 (qval < 1E-05)

mm9

Number of total reads
48453695
Reads aligned (%)
96.5
Duplicates removed (%)
22.1
Number of peaks
566 (qval < 1E-05)

Base call quality data from DBCLS SRA