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Sample GSM8282442

Query DataSets for GSM8282442

Status

Public on Jun 28, 2024

Title

10-Day idSCs r1

Sample type

SRA

Source name

Skeletal muscle in vitro derived satellite cell

Organism

Mus musculus

Characteristics

cell type: Skeletal muscle in vitro derived satellite cell

Growth protocol

Satellite cells refer to cells freshly isolated by FACS and immediately used, whereas primary myoblasts refer to cells derived from satellite cells, cultured in vitro for longer than two weeks, and used for experiments between passage 5-15. Satellite cells and primary myoblast lines were derived using hindlimb skeletal muscle from C57BL/6J, Tg:Pax7nGFP. Hindlimb skeletal muscles were aseptically dissected from either male or female 2-6 month old mice. For the derivation of primary myoblast lines, cells were cultured in GROWTH media: Ham’s F10 (Wisent), 20% heat inactivated FBS (Sigma Aldrich), 5ng/mL human bFGF, Aldevron), Non-Essential Amino Acids and GlutaMAX (Gibco) on collagen coated plates (Rat tail collagen, (Corning) at 37˚C with 5% CO2. Following culture ex vivo for 1 week, myoblasts were purified based on nGFP (Tg:Pax7nGFP) or α7 integrin (UBC Ablab). Purified myoblasts were passaged 5 times prior to being frozen as cell stocks. Differentiated myotubes or reserve cells were generated from proliferative myoblasts, grown to confluence, and subsequently cultured for 5 days in DIFFERENTIATION medium composed of DMEM (Gibco), 5% heat inactivated Horse Serum (Life Technologies), Non-Essential Amino Acids (Gibco) and Glutamax (Gibco) on laminin coated plates (Thermo Fisher). Mouse skeletal muscle organoids (SkMOs) were generated from myoblasts (passage 5-15) cultured in GROWTH medium on collagen coated plates. Cells were seeded in SPIN medium (DMEM:F12 (Gibco), 20% heat inactivated FBS (Sigma Aldrich), 10ng/mL human bFGF (Aldevron), Non-Essential Amino Acids and Glutamax (Gibco)). SkMOs were fed every 48hrs by allowing the organoids to settle, aspirating media, and adding 50mls of fresh SPIN medium. After 20 days, the medium was changed to DIFFERENTIATION medium for 10 days. SkMOs were cultured for 10 (D10), 20 (D20), or 30 (D30) days before isolating the in vitro derived satellite cells (idSCs). idSCs are defined as the SkMO-derived GFP+ cells.

Extracted molecule

genomic DNA

Extraction protocol

ATAC-seq experiments were conducted according to the Omni-ATAC protocol (Corces, M.R. et al. An improved ATAC-seq protocol reduces background and enables interrogation of frozen tissues. Nat Methods 14, 959-962 (2017).)
Library preparation was completed with 55ng input using a Nextera DNA kit (Illumina) following the manufacturer’s instructions

Library strategy

ATAC-seq

Library source

genomic

Library selection

other

Instrument model

Illumina NextSeq 500

Data processing

Basecalls were performed with bcl2fastq for NextSeq output.
Raw reads were trimmed of adapters using Ngmerge and aligned to mm10 using Bowtie2.
Mitochondrial reads were removed using removeChrom and PCR duplicates removed using Picard.
Peak calling was performed using Genrich. BAM files were sorted by name using SAMtools, and peaks were called using default ATAC parameters (-j -p 0.01 -a 200) and excluding the ENCODE blacklist for mm10. Consensus peaksets from replicates were called using IDR.
Assembly: mm10
Supplementary files format and content: bigWig, narrowPeak except for input files

Submission date

May 20, 2024

Last update date

Jun 28, 2024

Contact name

Kristina M Holton

E-mail(s)

kmholton@g.harvard.edu

Organization name

Harvard University

Department

HSCRB