GSM8684829: 12wk NT Rep 1, 12wk R62 Rep 1, 12wk NT Rep 2, 4wk R62 Rep 2 Mixed FLEX single nuclei-RNA-seq; Mus musculus; RNA-Seq
Fresh frozen tissue were OCT embedded, snap frozen over isopentane bath immersed in liquid nitrogen and ~700uM of 30uM sections of tissue were collected. Sections were fixed for 21 hours with 4% PFA and dissociated using Liberase TL (postnatal day 0) or Liberse TH (4-week and 12-week) and processed with the 10x Genomics Chromium Fixed RNA Profiling kit according to manufacter's instructions. Tissue was then dissoaciated with a gentleMACS OctoDissociator and cell quality and number evaluated. Probe hybiridization and library construction were performed with the 10x Genomics Single Cell Gene Expression FLEX according to the manufacter's instructions. 4 samples were multiplexed and 2 samples singleplex. After probe hybridization, nuclei were pooled (if multiplexed), partitioned into GEMs and ligation and extension was in GEMs  Samples were sequenced with the Illumina NovaSeq system with a target sequenciong depth of at least 30,000/nuclei.