title: GSM2629520: PAX7+ prolif_myog_rep5; Homo sapiens; RNA-Seq
library_construction_protocol: RNA-seq: Cells were resuspended in Trizol (Invitrogen) and RNAs were purified using the Purelink RNA mini kit (Life Technologies) following the manufacturer’s instructions. DNase treatment was performed on column during the RNA isolation procedure. ChIP-seq: Day 23 Proliferating Myogenic progenitors (+dox) were collected using trypsin and reaction was inhibited by adding 10%FBS/PBS. Single cells were washed once with PBS, resuspended in 10%FBS/PBS and supplemented with formaldehyde (final concentration 1%) for crosslinking of protein-DNA complexes (10 minutes at RT) followed by quenching with glycine. Cells were snap-frozen in liquid nitrogen and stored at -80°C if not processed immediately. RNA-seq: Sequencing libraries were generated from 100 ng of total RNA using the Ligation Mediated Sequencing (LM-Seq) protocol (Hou et al., 2015) quantitated using the Qubit fluorometer (Life Technologies) following the author’s instructions. ChIP-seq: Libraries were generated following a gel-free protocol using AMPure XP beads (Beckman Coulter) for all the purification and size selection steps. 10ng or less of DNA were end repaired using End-it DNA end repair (Epicentre), then A-tailed using Klenow Fragment (3'→5' exo-  NEB) followed by adapter-barcode ligation using T4 DNA ligase (Enzymatics). Illumina compatible adapter-barcodes were purchased from BIOO scientific. After ligation, DNAs were negatively size selected using 0.5x Ampure XP beads and unbound DNAs were positively size selected by adding 0.4x Ampure XP beads (this step allows for retention of DNA fragments ranging 200-500bp). Libraries were amplified using Phusion High Fidelity PCR master mix 2x (NEB) with a 16 cycles program.
attributes.tag: GEO Accession
attributes.value: GSM2629520