title: GSM7865617: Heart, Control_NIL, snRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: Heart tissue samples were collected  and minced. Minced samples were resuspended in 5 mL Lysis Buffer (320 mM sucrose, 10 mM Tris-HCl, 5 mM CaCl2 , 5 mM Magnesium Acetate, 2 mM EDTA, 0.5 mM EGTA, 1 mM DTT, 1x complete protease inhibitor (Roche), 200U/mL RNase OUT Recombinant Ribonuclease Inhibitor (Invitrogen, 10777-019), pH=8) and homogenized with a dounce grinder for 15 strokes. The lysate was sequentially ﬁltered through 70 um and 40 um cell strainers (Falcon, 352350 and 352340) and centrifuged at 1,000 x g for 5 min at 4℃ to pellet nuclei. The nuclear pellet was subsequently resuspended in 2 mL Sucrose Buffer (1 M sucrose, 10 mM Tris-HCl, 5 mM magnesium acetate, 1mM DTT, 1x complete protease inhibitor, 200 U/mL RNase OUT Recombinant Ribonuclease Inhibitor, pH=8) and the suspension was cushioned on top of 4 mL Sucrose Buffer, followed by centrifugation at 1,000 x g for 5 min at 4℃ to pellet nuclei. The nuclear pellet was subsequently washed once in 1 mL of Nuclei Storage Buffer (NSB) (440 mM sucrose, 10 mM Tris-HCl, 70 mM KCl, 10 mM MgCl2 , 1.5 mM spermine, 1x complete protease inhibitor, 200 U/mL RNase OUT Recombinant Ribonuclease Inhibitor, pH=7.2). Single nucleus RNA-seq libraries were generated using Single Cell 3'Reagent Kits v2 (10xGenomics) according to the manufacturer's protocol. Brieﬂy, cells, reagents (10xGenomics, PN-120237), gel beads (10xGenomics, 220104), and partitioning oil (10xGenomics, 220088) were loaded into the Chromium Single Cell A Chip (10xGenomics, 2000019), targeting 2000、5000 nuclei to be recovered per channel. Chip was loaded to the Chromium Controller (10xGenomics) to generate Gel Bead-In-Emulsions (GEMs). After reverse transcription using GEM-RT incubation protocol, GEMs were broken with recovery reagent (10xGenomics, 220016), cDNA was puriﬁed with DynaBeads MyOne Silane beads (Thermo Scientiﬁc, 37002D), ampliﬁed with PCR for 14 cycles, and further puriﬁed with SPRIselect reagent (Beckman Coulter, B23318). Quality control of recovered PCR product was performed on a Tapestation (Agilent) with D5000 high sensitivity screentapes (Agilent, 5067-5592), and cDNA yield was measured by Qubit DNA high sensitivity assay (Invitrogen, Q32854). After fragmentation, end-repair, A-tailing and size selection puriﬁcation, cDNA was ligated with adaptor and subsequently puriﬁed with SPRIselect reagents. Libraries were ampliﬁed using PCR with sample indexes from Chromium i7 Multiplex kit (10xGenomics, PN-120262). Final PCR products were subjected to double-sided size selection with SPRIselect reagents. Library quality control was performed on the Agilent Tapestation with D1000 screentapes (Agilent, 5067-5582), and library yield was quantiﬁed by Qubit DNA high sensitivity assay. Sequencing was performed on an Illumina NovaSeq 6000 system.