GSM4654471: Nuc-RM77-2; Homo sapiens; RNA-Seq
Brain tissue was sliced on dry ice, then homogenized manually (15 gentle strokes) with 1mL ice-cold  Homogenisation Buffer (HB) with 5µL Rnasin Plus. The homogenate was strained with a 70µm strainer and washed with 1.65mL to a final volume of 2.65mL. 2.65mL of Gradient medium was added (Vf = 5.3mL). To isolate the nuclei, the sample was added to a 4mL 29% cushion using a P1000, and the weight adjusted with HB. The sample was centrifuged in a SW41Ti rotor at 7,700 rpm for 30 minutes at 4°C. The supernatant was removed with a plastic Pasteur pipette, followed by removal of the lower supernatant with P200. Nuclei were resuspended in 200µL of resuspension buffer, transferred to a new tube, washed again with 100-200µL resuspension buffer, and pooled with the previous solution. Clumps were disrupted by pipetting with P200, then filtered through a Falcon tube with 0.35µm strainer. 9µL of sample was mixed with 1µL of propidium iodide (PI) stain, loaded onto a LUNA-FL slide and allowed to settle for 30 seconds. We viewed nuclei with the LUNA-FL Automated cell counter to check numbers and shape. RNA sequencing was performed using the 10x Genomics Single Cell 3` Reagent Kit (v2) according to manufacturer protocols. cDNA libraries from fresh-frozen nuclei were sequenced on an Illumina HiSeq platform 4000.