Antigen

Antigen Class

Input control

Antigen

Input control

Cell type

Cell type Class

Muscle

Cell type

Myoblasts

MeSH Description

Embryonic (precursor) cells of the myogenic lineage that develop from the MESODERM. They undergo proliferation, migrate to their various sites, and then differentiate into the appropriate form of myocytes (MYOCYTES, SKELETAL; MYOCYTES, CARDIAC; MYOCYTES, SMOOTH MUSCLE).

Sample

source_name

human immortalized myoblasts

cell type

unaffected human myoblasts

antibody

none

Sequenced DNA Library

library_strategy

ChIP-Seq

library_source

GENOMIC

library_selection

ChIP

library_construction_protocol

Cell fixation was performed 24 hours post-transfection by the addition of 1% formaldehyde for 15 min and quenched with 0.125 M glycine. Chromatin was isolated by the addition of lysis buffer, followed by disruption with a Dounce homogenizer. Lysates were sonicated and the DNA sheared to an average length of 300-500 bp. Genomic DNA (Input) was prepared by treating aliquots of chromatin with RNase, proteinase K and heat for de-crosslinking, followed by ethanol precipitation. Pellets were resuspended and the resulting DNA was quantified on a NanoDrop spectrophotometer. An aliquot of chromatin (30 ug) was precleared with protein A agarose beads (Invitrogen). Genomic DNA regions of interest were isolated using 4 µg of antibody against V5-tag (Abcam ab15828). Complexes were washed, eluted from the beads with SDS buffer then subjected to RNase and proteinase K treatment. Crosslinks were reversed by incubation overnight at 65 °C, and ChIP DNA was purified by phenol-chloroform extraction and ethanol precipitation. Illumina sequencing libraries were prepared from the ChIP and Input DNA by the standard consecutive enzymatic steps of end-polishing, dA-addition, and adaptor ligation. After a final PCR amplification step, the resulting DNA libraries were quantified and sequenced on HiSeq 2500. Sequences (50 nt reads) were aligned to the human genome (hg19) using the BWA algorithm.

Sequencing Platform

instrument_model

Illumina HiSeq 2000

hg38

Number of total reads

31092402

Reads aligned (%)

92.1

Duplicates removed (%)

5.1

Number of peaks

1333 (qval < 1E-05)

hg19

Number of total reads

31092402

Reads aligned (%)

91.3

Duplicates removed (%)

6.5

Number of peaks

1265 (qval < 1E-05)