GSM9136561: CTRL_4; Homo sapiens; RNA-Seq
Nuclei were isolated from frozen postmortem tissues as previously described with modifications. The tissues were homogenised in chilled lysis buffer (10 mM Tris, 10 mM NaCl, 3 mM MgCl2, 0.05% NP40) using a glass Dounce homogeniser, with 10 loose pestle strokes, followed by 5 tight pestle strokes. The homogenates were filtered through a 40 μm strainer and centrifuged for 5 min at 500 × g, 4°C to pellet the nuclear fraction. The pellet was then resuspended in 10 ml of wash buffer (5% BSA, 40 units/ml Protector RNase inhibitor in 0.5× PBS) and centrifuged for 5 min at 500 × g, 4°C. The pellet was then resuspended in 1 ml wash buffer, mixed with an equal volume of 50% OptiPrep solution, and layered on top of 2 ml of 29% OptiPrep solution. The nuclei were separated by centrifugation at 1,000 × g for 30 min at 4°C. The nuclear pellet was washed three times with wash buffer (2% BSA, 40 units/ml Protector RNase inhibitor in 0.5× PBS) and subsequently resuspended in 500 μl wash buffer to yield a clean single-nucleus suspension. For quantification, the collected nuclei were stained with DAPI and counted with Countess II Automated Cell Counter (Life Technologies). An estimated 10,000 human nuclei were processed using the Chromium Single Cell 3′ v3 Gene Expression Reagent Kits, following the manufacturer's instructions (10x Genomics). The generated snRNA-seq libraries were sequenced using an Illumina HiSeq 4000 at NovogeneAIT Genomics Singapore Pte Ltd.