Sample information curated by ChIP-Atlas

Antigen

Antigen Class
TFs and others
Antigen
Pax7

Cell type

Cell type Class
Muscle
Cell type
Myogenic progenitors
NA
NA

Attributes by original data submitter

Sample

source_name
Pax7 in iPax7 myogenic progenitors +dox
treatment
0.75ug/ml dox in iPax7 media
chip antibody
Pax7 (DSHB, 3 ug for ChIP)

Sequenced DNA Library

library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
[ATAC-seq] Cells were harvested by trypsinization and resuspended in appropriate media. 500,000 cells were subsequently used for nuclear extraction, except for satellite cells (described separately) and myotubes where 100,000 cells were used to adjust for their multinucleated nature. Cells were washed with 500 µl cold PBS and resuspended in 500 µl cold lysis buffer (10mM Tris-HCl pH 7.4, 10mM NaCl, 3mM MgCl2, 0.1% IGEPAL CA-630), and spun at 500g for 10 minutes at 4°C. Supernatant was discarded and the nuclear pellet was resuspended in nuclease-free water and 50,000 cells (10,000 cells for myotubes) in 22.5 µl nuclease free water was mixed with 25 µl TD Buffer and 2.5 µl Tn5 Transposase (Illumina cat #FC-121-130). 50,000 freshly sorted satellite cells from Pax7-ZsGreen mice were washed with 200 µl of cold PBS then resuspended in 100 µl of cold lysis buffer, spun at 500 g for 10 minutes at 4°C and resuspended in 50 µl of the transposition reaction mix. Transposition occurred at 37°C for 30 minutes, after which transposed DNA was purified using a Qiagen MinElute Kit and eluted in 10 ml Elution Buffer. Transposed DNA fragments were mixed with 7 μl nuclease free H2O, 2.5 μl of each forward and reverse 25 µM stock of Nextera PCR primers, 3 μl of 1000x diluted stock of SYBR Green I (Invitrogen #S-7563), 25 μl NEBNext High-Fidelity 2x PCR Master Mix (New England BioLab #M0541). All samples were amplified for 5 cycles (as described in (Buenrostro et al., 2013)) after which 5 µl was aliquoted to determine optimal cycle numbers. 5 μl was mixed with 1.9 μl Nuclease Free H2O, 1.25 μl of each forward and reverse customized 5 μM stock of Nextera PCR primers, 0.6 µl of 1000x diluted stock of SYBR Green I, and 5 μl NEBNext High-Fidelity 2x PCR Master Mix. All samples were amplified for 20 cycles, and the number of additional cycles to the remaining transposed DNA was determined as described by (Buenrostro et al., 2013). Libraries were then purified with 1.2x vol AMPure (Beckman) beads. Tagmentation was confirmed via Tapestation. [RNA-seq] Bioanalyzer RNA quality and quantity assay picochip was used to measure RNA concentrations for each of the collected samples and ca 1.5 ng/sample were used to generate RNA-seq libraries with SMARTer® Ultra® Low Input RNA for Illumina® Sequencing – HV (Clontech Laboratories) using the manufacturer’s instructions. Libraries were sequenced (51 bp, paired end) on an Illumina HiSeq 2500 machine with v4 chemistry. RNA-seq libraries were prepared with the manufacturer’s instructions. [ChIP-seq] ChIP and ChIP-seq libraries were prepared as described (Asp, P., Blum, R., Vethantham, V., Parisi, F., Micsinai, M., Cheng, J., . . . Dynlacht, B. D. (2011). Genome-wide remodeling of the epigenetic landscape during myogenic differentiation. Proc Natl Acad Sci U S A, 108(22), E149-158. doi:10.1073/pnas.1102223108) and sequenced (51 bp, single reads) using an Illumina HiSeq 2500 machine.

Sequencing Platform

instrument_model
Illumina HiSeq 2500

mm10

Number of total reads
80857789
Reads aligned (%)
78.4
Duplicates removed (%)
19.1
Number of peaks
1781 (qval < 1E-05)

mm9

Number of total reads
80857789
Reads aligned (%)
78.3
Duplicates removed (%)
19.0
Number of peaks
1883 (qval < 1E-05)

Base call quality data from DBCLS SRA