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Sample GSM2629506

Query DataSets for GSM2629506

Status

Public on Jun 30, 2017

Title

mesod_nodox_rep1

Sample type

SRA

Source name

Unsorted cells collected at day 14

Organism

Homo sapiens

Characteristics

cell type: H9 cells
doxycycline-treatment: untreated
collection time: day 14 of differentiation

Growth protocol

Inducible PAX7 H9 ES cells were plated in 6cm non-adherent Petri dishes and incubated at 37°C, 5% CO2 on a shaker at 60 RPM. After 2 days, mTeSR was replaced with EB Myogenic (EBM) Media supplemented with 10µM Y-27632 and 5µM GSK3 inhibitor (CHIR990217). 2 days later, media was replaced to remove GSK3 inhibitor and the formed EBs were cultured in suspension on the shaker up to day 7. After plating EBs on gelatin-coated flasks using EBM + 10ng/ml human basic FGF (bFGF), EBs were let to adhere on the flasks for 3 days and then supplemented with 1µg/ml doxycycline (dox) for PAX7 induction. At day 14, cells were harvested using Trypsin+EDTA solution and FACS sorted based on GFP expression. GFP+ cells were then plated at 1.5*10^6 cells per T25 on gelatin-coated flasks using EBM supplemented with 5ng/ml bFGF, 1µg/ml dox and 5µM ROCK inhibitor (ROCK inhibitor was removed the day after) and cultured as monolayer. At ~90% cell density, cells were passaged using Trypsin+EDTA and replated on new gelatin-coated flasks. Terminal differentiation was induced by switching 100% confluent cultures to KOSR differentiation media (KnockOut™ DMEM + 20% KnockOut™ Serum Replacement + 1% Penicillin/Streptomycin + 2mM GlutaMAX™ - all products from Gibco).

Extracted molecule

total RNA

Extraction 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.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2500

Description

s46154.00405

Data processing

Basecalls performed using CASAVA version 1.8.2
Reads filtered and trimmed to eliminate low-quality reads and adapter sequences
RNA-seq: Gene expression estimates obtained using RSEM v 1.2.3, with Bowtie 0.12.9 for alignment. Parameters to rsem-calc-expression include: "--bowtie-m 200 --bowtie-n 2 --forward-prob 0.5 --seed-length 28 ".
ChIP-seq: reads were then aligned to the human genome (hg38) using Bowtie2 followed by removal of PCR duplicates using the SAM tool rmdup 1.0.1 . Peak calling was performed using MACS 1.0.1 (parameters: -s 51 -bw 300, -p 1e-05 -m 16). Bigwig files for visualization on IGV were generated by converting the wig files obtained from MACS using the tool wigtoBigWig.
Genome_build: RNA-seq: hg19. ChIP-seq: hg38
Supplementary_files_format_and_content: RNA-seq: Excel .xlsx file containing TPM expression estimates calculated by RSEM.
Supplementary_files_format_and_content: ChIP-seq: BigWig files of Input and ChIP samples and list of PAX7-bound peaks in .bed format after removal of overlapping peaks detected in the Input and the human blacklist dataset

Submission date

May 16, 2017

Last update date

May 15, 2019

Contact name

Alessandro Magli

E-mail(s)

alemagli@gmail.com

Organization name

University of Minnesota

Department

Medicine