title: GSM8163686: Day2_SatelliteCells_Control_biol_rep1_ATAC; Mus musculus; ATAC-seq
library_construction_protocol: RNA-seq: At least 10,000 FACS sorted muscle stem cells were deposited into Trizol (Thermo Fisher) and snap frozen immediately thereafter. After thawing, total RNA was isolated using the miRNeasy Micro Kit (Qiagen) as per the manufacturer's instructions. The isolated RNA was quantitated and the integrity of the samples were verified with a Bioanalyzer (Agilent 2100). RNA-seq:  Approximately 10ng of isolated total RNA from biological replicates was used to produce cDNA libraries using the SmartSeq4 protocol (Clontech), as per the manufacturer's instructions. Individual libraries were pooled and sequenced using one lane of 76-bp paired-end reads on an Illumina High-Seq 2500 to an average depth of 50M reads per library. ATAC-seq: At least 10,000 sorted muscle stem cells were sorted into 1.5 mL centrifuge tubes, spun down at 500xg for 5 min at 4˚C and the supernatant removed. After re-suspension in cold 1x PBS and centrifugation again at 500xg for 5 min at 4˚C, the cell pellets were re-suspended in 50 µL of cold lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-360), and centrifuged at 750xg for 15 min at 4˚C to pellet the nuclei. The nuclei were then re-suspended transposase reaction mix (12.5 µL 2x TD buffer, 1.25 µL TN5 transposase (Illumina) and 11.25 µL nuclease-free water), incubated at 37˚C for 35 min under mild agitation (300 rpm on a Thermo-Mixer C, Eppendorf). The transposed DNA fragments were purified (Qiagen MinElute PCR Purification Kit) as per manufacturer's instructions, amplified using barcoded PCR primers and NEB Next High Fidelity 2x PCR Master Mix (New England Bio Labs) on a thermal cycler and purified again using double concentrated SPRI beads. ATAC-seq: The concentration and size distribution of each was determined (Agilent 2100 Bioanalyzer), pooled and sequenced on a NextSeq (Illumina) using 76-bp paired-end reads to greater than 50M reads per library.