snRNA of human pancreas: nPOD 6247
Roughly 40 mg ground pancreas tissue was suspended in 500 L of nuclei buffer. Sample was incubated on a rotator for 5 min at 4 C and then pelleted with a swinging bucket centrifuge (500 g, 5 min, 4 C; 5920 R, Eppendorf). Supernatant was removed and pellet was resuspended in 400 l of sort buffer and stained with DRAQ7 (1:100; Cell Signaling Technology, 7406). 75,000 nuclei were sorted using an SH800 sorter (Sony) into 50 l of collection buffer. Sorted nuclei were then centrifuged at 1000g for 15 min (Eppendorf, 5920R; 4C, ramp speed of 3/3), and supernatant was removed. Nuclei were resuspended in 18 to 25 l of reaction buffer [RNase inhibitor (0.2 U/l) and 1% BSA (Sigma-Aldrich, SRE0036) in PBS] and counted using a hemocytometer. 16,500 nuclei were loaded onto a Chromium controller (10x Genomics). Libraries were generated using the 10x Genomics, Chromium Next GEM Single Cell 3 GEM, Library & Gel Bead Kit v3.1 (10x Genomics, 1000121), Chromium Next GEM Chip G Single Cell Kit (10x Genomics, 1000120) and indexes (Single Index Kit T Set A, 10x Genomics, 1000213 or Dual Index Kit TT Set A, 10x Genomics, 1000215) 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 a TapeStation (High Sensitivity D1000, Agilent). Libraries were sequenced using a Novaseq6000 (Illumina) with the following read lengths (Read1 + Index1 + Index2 + Read2): 28 + 8 + 0 + 90 (single index) or 28 + 10 + 10 + 90 (dual index).