GSM7731331: KI990s34_heart_nuclei; Mus musculus; RNA-Seq
Mice were anesthetized with isoflurane, euthanized by cervical dislocation, and then hearts were perfused with 100 mM KCl. Removed hearts were diced into small pieces in cold PBS containing Diethyl pyrocarbonate (DEPC, Sigma D5758). Fresh heart pieces were dounce-homogenized to liberate nuclei in Hypotonic lysis buffer B (7.7 mM Na2HPO4, 4.5 mM NaH2PO4, 1.8 mM KH2PO4, 2.7 mM KCl, 10.3 mM NaCl, 3 mM MgCl2, 0.08% BSA, 0.025% Igepal CA-630, 1% DEPC) and passed through cell strainers. Nuclei were fixed in 1 mg/mL dithiobis(succinimidyl propionate) (DSP, Thermo Fisher 22585) for 15 min on ice, washed, and stored at –80°C until use. For each mouse, 50,000 nuclei were placed in each of 8 wells of a 96-well plate (total 48 wells for 6 mice). The remaining 48 wells were filled with similarly processed mouse heart nuclei with comparable sample quality from an unrelated project. Nuclei were subjected to reverse-transcription with 96-indexed poly-T RT primers that contained a unique molecular identifier (UMI) sequence. Nuclei from all wells were combined and redistributed evenly to 96 wells. Redistributed cells were subjected to ligation with 96-indexed ligation primers in 96 wells (Table SX). Nuclei from all wells were combined again and redistributed evenly to 96 wells at 1,000 nuclei per well. Redistributed cells underwent second strand synthesis, protease digestion, tagmentation using N7-loaded Tn5 transposase, and PCR-amplified using 96-indexed PCR P7 primers and the unindexed P5 primer, for 16 cycles. PCR amplicons were combined and size-selected for DNA fragments between 250 bp and 600 bp. Combinatorial indexing-based single-nucleus RNA-seq (sci-RNA-seq3)