GSM9431046: snRNA-seq library from donor A.4; Homo sapiens; RNA-Seq
Nuclei were isolated from frozen retinal tissue on ice using the Nuclei EZ Prep Kit (NUC-101, Sigma-Aldrich) with minor modifications. All steps were performed on ice or at 4 °C. Tissue was dounce-homogenized (25 strokes with loose pestle A followed by 25 strokes with tight pestle B) in 2 mL EZ Lysis Buffer using a KIMBLE Dounce Tissue Grinder. The homogenate was transferred to a 15 mL tube, supplemented with an additional 2 mL EZ Lysis Buffer, incubated on ice for 5 min, and centrifuged at 500 × g for 5 min at 4 °C. The supernatant was discarded, and the nuclei pellet was resuspended in 4 mL EZ Lysis Buffer, incubated on ice for 5 min, and centrifuged again at 500 × g for 5 min at 4 °C. Nuclei were washed in 4 mL ice-cold Nuclei Suspension Buffer (1× PBS containing 0.01% BSA and 0.1% RNase inhibitor), resuspended in 1 mL Nuclei EZ Storage Buffer, and filtered through a 40 µm nylon cell strainer. Nuclei were counted using trypan blue exclusion with a hemocytometer prior to library preparation. RNA libraries of barcoded single nuclei were generated using the 10x Genomics Chromium Next GEM Single Cell 3′ Library & Gel Bead Kit according to the manufacturer's instructions. Briefly, isolated nuclei were loaded onto the Chromium Controller, where individual nuclei were partitioned into Gel Beads-in-Emulsion (GEMs) for barcoding. Within each GEM, reverse transcription was performed to generate barcoded cDNA molecules containing unique cell barcodes and unique molecular identifiers (UMIs). Following GEM cleanup and cDNA amplification, sequencing libraries were constructed through fragmentation, end repair, A-tailing, adaptor ligation, and sample index PCR. Final libraries were quantified and assessed for quality prior to sequencing.