GSM9514914: Young Sham3; Mus musculus; RNA-Seq
Total RNA was extracted via Trizol-Chloroform precpitiation of RNA using Zymo Direct-Zol RNA extraction kit. Genomic DNA was extracted from myonuclei isolated from the plantaris muscle via fluorescent activated nuclear sorting. The Quick-DNA™ Miniprep Plus kit (Zymo Research, D4068) was used as specified in the protocol. For single myonucleus RNA-seq, myonuclei were isolated from plantaris muscle chunks were minced with scissors in homogenization buffer (500 µl HEPES (1 M) 3 ml KCl (1 M) 250 µl spermidine (100 mM) 750 µl spermine tetrahydrochloride (10 mM) 10 ml EDTA (10 mM) 250 µl EGTA (100 mM) 2.5 ml MgCl (100 mM) 5.13 g sucrose, Dounce homogenized on ice with a plastic pestle in homogenization buffer, then passed through a 40 µm and 20 µm filter into directly into the MACSQuant High Speed Tyto Cardtridge. PI was added to label nuclei. Fluorescently labeled nuclei (GFP+/PI+) were purified via Fluorescence-activated nuclear sorting (FANS) and sorted into re-suspension buffer (1X PBS, 1% BSA, 0.2U/uL RNase inhibitors). Total RNA-seq libraries were prepared for seqeuencing using standard Illumina protocols and underwent 150 bp PE sequencing. The DNA RRBS was rered to Zymo Research Corporation (RRBS Basic Srvise Package, 15-20 million read pairs per sample). Myouclei were mixed with reverse transcriptase buffer and loaded into the 10X Chromium system using the Single Cell 3' Reagent Kit v4 according to the manufacturer's protocol. Following library construction, libraries were sequenced on the Illumina NovaSeq X Plus System at the Novogene.