title: GSM7246725: Hypertrophied AT, snRNA seq; Homo sapiens; RNA-Seq
library_construction_protocol: Frozen biopsy samples were transferred into 1.5 mL microcentrifuge tubes on ice to which 50 μL of nuclei extraction buffer (NEB) (10 mM HEPES, 1.5 mM MgCl2, 10 mM KCl, 250 mM sucrose, 0.1% NP-40, and 0.2 U/μL RNase inhibitor [Sigma, 3335399001]) were added. Sterile 0.5 mL microcentrifuge tubes were fitted into the 1.5 mL microcentrifuge tubes, allowing samples to be homogenized with 12 strokes with rotation, while maintaining the samples on ice. After addition of 3 mL of NEB, samples were incubated on ice for 1 min with gentle mixing followed by centrifugation at 1000 x g for 10 min at 4°C. With the supernatant removed, the nuclei pellet was resuspended in 500 μL of nuclei resuspension buffer (NRB) (0.5% BSA, 0.2 U/μL RNase inhibitor [Sigma, 3335399001], and 20 μg/mL DAPI [Fisher, 62248] in PBS). The nuclei suspension was then filtered through a 55 μm nylon mesh and observed under a fluorescence microscope for nuclei integrity and absence of clumps. The filtered suspension was then purified using FACS sorting (BD FACS Aria III, BD, Franklin Lakes) for DAPI+ nuclei. All plasticware had been coated with 4% BSA in PBS overnight at 4°C. Single nuclei were captured using Chromium Next GEM Single Cell 5' v2 according to the manufacturer's instructions (10X Genomics, Pleasanton). Around 10000 nuclei were loaded into the reaction chip. Sequencing was done with NextSeq 500/550 High Output Kit v2 aiming at a target depth of 20000 reads/nucleus, with read lengths of 26 cycles (read 1), 10 cycles (i7 index), 10 cycles (i5 index), and 120 cycles (read 2).