GSM6398368: TB5053; Homo sapiens; RNA-Seq
Briefly, frozen tissue samples measuring from 5 x 4 x 3 mm to 10 x 6 x 3 mm were homogenized using a dounce homogenizer in ice-cold 30% sucrose 0.1% Triton-X 100 based homogenization buffer. 10-15 strokes of the loose dounce pestle were followed by 10-15 strokes of the tight dounce pestle on ice. Mixing using a P1000 pipette followed before filtration through a BD Falcon 40um filters.  Filtration was repeated after a 10-minute spin at 1000g at 4c. A cleanup step followed using a density gradient step as described in [41]. The nuclear pellet was suspended in 1% BSA in PBS resuspension buffer containing RNAse inhibitors. A final filtration step using 20um Flowmi ™ filters followed before dilution to 700-1200 nuclei per ul in resuspension buffer. The 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, catalog number: 120237; Chromium Single Cell A Chip Kit, 48 runs, catalog number: 120236; 10x Genomics). Sequencing of the resultant libraries was done on Illumina NOVAseq 6000 platformV4 150bp paired end reads.