GSM6123263: MM_466_S15; Homo sapiens; RNA-Seq
Approximately ~35 mg of frozen pancreas from each donor was resuspended in 500 μl of nuclei permeabilization buffer [0.1% Triton X-100 (Sigma-Aldrich, T8787), 1X Pierce Protease Inhibitor (Fischer, PIA32965), 1 mM DTT (Sigma-Aldrich, D9779), Recombinant RNase inhibitor (0.2 U/μl; Promega, 2% Fatty-acid-free BSA in PBS (Proliant, 7500804; Corning, 21-040-CV)]. Sample was incubated on a rotator for 5 min at 4°C and then centrifuged at 500g for 5 min (Eppendorf, 5920R; 4°C, ramp speed of 3/3). Supernatant was removed and pellet was resuspended in sort buffer [1mM EDTA (Invitrogen, 15575020), 0.2U/μL Recombinant RNAsin (Promega, PAN2515), 1% Fatty-acid-free BSA in PBS (Proliant, 7500804; Corning, 21-040-CV)).] and stained with DRAQ7 (1:150; Cell Signaling Technology, 7406). 60,000 nuclei were sorted using an SH800 sorter (Sony) into 50 μl of collection buffer [1.0U/μL Recombinant RNAsin (Promega, PAN2515), 5% Fatty-acid-free BSA in PBS (Proliant, 7500804; Corning, 21-040-CV)]. Sorted nuclei were then centrifuged at 1000 g for 15 min (Eppendorf, 5920R; 4°C, ramp speed of 3/3), and supernatant was removed. Nuclei were resuspended in reaction buffer [RNase inhibitor (0.2U/μL Recombinant RNAsin (Promega, PAN2515), 1% Fatty-acid-free BSA in PBS (Proliant, 7500804; Corning, 21-040-CV)).] and counted using a hemocytometer. 16,550 nuclei were loaded onto a Chromium controller (10x Genomics). Libraries were generated using the Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1 (10x Genomics, PN-1000121) according to the manufacturer specifications. Complementary DNA was amplified for 12 PCR cycles. SPRISelect reagent (Beckman Coulter) was used for size selection and cleanup steps. Final library concentration was assessed by the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific), and fragment size was checked using TapeStation High Sensitivity D1000 (Agilent) to ensure that fragment sizes were distributed normally around 500 bp. Libraries were sequenced using a NextSeq 500 or NovaSeq 6000 (Illumina). Single nucleus RNA-seq with Chromium Next GEM Single Cell 3' GEM, Library & Gel Bead Kit v3.1 (10x Genomics)