GSM7680727: 8035073605-N, Circadian control, GEX, bio rep 2; Homo sapiens; RNA-Seq
Frozen human skeletal muscle samples were pulverized and the powder used for nuclei isolation. Briefly, and all on ice, RNAse inhibitor (NEB cat# MO314L) was added to the homogenization buffer (0.32 M sucrose, 1 mM EDTA, 10 mM Tris-HCl, pH 7.4, 5mM CaCl2, 3mM Mg(Ac)2, 0.1% IGEPAL CA-630), 50% OptiPrep (Stock is 60% Media from StemCell cat# 07820), 35% OptiPrep and 30% OptiPrep right before isolation. Each sample was homogenized in a Dounce glass homogenizer (1ml, VWR cat# 71000-514), and the homogenate filtered through a 40 mm cell strainer. An equal volume of 50% OptiPrep was added, and the gradient centrifuged (SW41 rotor at 17,792xg; 4C; 25min). Nuclei were collected from the interphase, washed, resuspended in 1X nuclei dilution buffer for sn multiome (10x Genomics) and counted (Cellometer). Sn multiome was performed following the Chromium Single Cell Multiome ATAC and Gene Expression Reagent Kits V1 User Guide (10x Genomics, Pleasanton, CA). Nuclei were counted (Countess counter), transposition was performed in 10 ul at 37C for 60min targeting up to 10,000 nuclei, before loading of the Chromium Chip J (PN-2000264) for GEM generation and barcoding. Following post-GEM cleanup, libraries were pre-amplified by PCR, after which the sample was split into three parts: one part for generating the snRNAseq library, one part for the snATACseq library, and the rest was kept at -20C. SnATAC and snRNA libraries were indexed for multiplexing (Chromium i7 Sample Index N, Set A kit PN-3000262, and Chromium i7 Sample Index TT, Set A kit PN-3000431 respectively). Single Cell Multiome ATAC and Gene Expression