GSM4140579: MuSC d0 Young bulkATACseq Rep2; Mus musculus; ATAC-seq
Sample information curated by ChIP-Atlas
Antigen
Antigen Class
ATAC-Seq
Antigen
ATAC-Seq
Cell type
Cell type Class
Muscle
Cell type
Muscle stem cells
NA
NA
Attributes by original data submitter
Sample
source_name
MuSCs, Pre-injury, Young
cell type
Flow sorted muscle stem cells
age
Young
timecourse
Day0
Sequenced DNA Library
library_strategy
ATAC-seq
library_source
GENOMIC
library_selection
other
library_construction_protocol
Hind limb muscles (tibialis anterior and gastrocnemius) of control and experimental mice were dissected using sterile surgical tools, tissues were minced and transferred into 50 mL conical tubes containing 20 mL of digest solution. Cell sorting was done using a BD FACSAria III Cell Sorter. Chromatin Accessibility Assay and Sequencing Library Preparation. At least 10,000 MuSCs were centrifuged at 500xg for 5 min at 4˚C in a fixed angle centrifuge. After removing the supernatant, the cell pellets were re-suspended in 50uL ATAC-Resuspension Buffer (RSB) (500uL 1M Tris-HCl, 100uL 5M NaCl, 150uL 1M MgCl2 in 49.25mL sterile water) with 0.1% NP40, 0.1% tween-20, and 0.01% Digitonin. Samples were pipetted up and down three times to mix, incubated 3 minutes on ice, then diluted in 1mL of ice-cold ATAC-RSB containing 0.1% Tween-20 and centrifuged at 500xg for 10 min at 4˚C to pellet the nuclei. The supernatants were carefully removed and the pellets were re-suspended in 25 µL of transposase reaction mix (12.5 µL 2x TD buffer, 1.25 µL TN5 transposase (Illumina) and 11.25 µL nuclease-free water). Transposase reactions were carried out by incubating samples at 37˚C for 30 min under mild agitation (300 rpm on a Thermo-mixer C, Eppendorf). Once the incubation was completed, sample tubes were placed on ice and the transposed DNA fragments from each sample were purified using a Qiagen MinElute PCR Purification Kit following manufacture's protocol. Purified DNA fragments were then amplified for 13 cycles using barcoded PCR primers and NEB Next High Fidelity 2x PCR Master Mix (New England Bio Labs) on a thermal cycler. Double concentrated Ampure beads were used to purify transposed DNA amplicons. The molarity of each DNA library was determined (Agilent 2100 Bioanalyzer), pooled into a single tube and sequenced on a NextSeq (Illumina) using 76-bp paired-end reads. Bulk mRNA Isolation and Sequencing Library Preparation. MuSCs sorted directly into Trizol were thawed at room temperature, and RNA was extracted using a Qiagen miRNeasy Micro Kit as per manufacturer's instructions. RNA concentration and integrity were measured with a Nanodrop spectrophotometer (Nanodrop 2000c) and Bioanalyzer (Agilent 2100). 1-10 ng of high-quality RNA (RIN>8) was used to produce cDNA libraries using the SmartSeq v4 protocol (Clontech) as per the manufacturer's instructions. cDNAs were prepared into sequencing libraries using 150 pg of full-length cDNA amplicons (Nextera XT DNA Library Preparation Kit, Illumina) with dual index barcodes as per manufacturer's instructions. Barcoded cDNA libraries were pooled into a single tube and sequenced on a NextSeq (Illumina) using 76-bp single-ended reads.