GSM7754789: H5575Cd; Homo sapiens; RNA-Seq
The tissue was homogenized in a Dounce homogenizer with 12–15 strokes of the loose pestle and 12–15 strokes of the tight pestle on ice in a Triton X-100 based, sucrose containing buffer. The suspension from each sample was filtered through a BD Falcon tubes with a cell strainer caps (Becton Dickinson, cat. no. 352235), washed, re-filtered, washed, followed by a cleanup step using iodixanol gradient centrifugation. The nuclear pellet was then re-suspended in 1% BSA in 545 nuclease-free PBS (containing RNAse inhibitors) and titrated to 600-1200 nuclei/μl. Nuclear suspensions were processed by the Chromium Controller (10x Genomics) using single Cell 3′ Reagent Kit v2 or v3 (Chromium Single Cell 3′ Library & Gel Bead Kit v2/v3, catalog number PN-1000075; Chromium Single Cell A Chip Kit, 48 runs, catalog number: 120236; 10x Genomics). Sequencing and alignment: Sequencing of the snRNAseq libraries was done on Illumina NOVAseq 6000 platformV4 150 bp paired end reads. snRNA (single-nuclei RNA sequencing)