GSM8970404: Pancreatic islet cells, CTRL (biol rep 2); Mus musculus; RNA-Seq
Tthe main pancreatic duct was injected with 2 mL ice-cold 1x HBSS containing 0.140 mL CIzyme RI (PELOBiotech, VitaCyte, 005-1030). The pancreas was digested for 17 min. at 37°C in 1 mL HBSS supplemented with 7.5% (w/v) sodium bicarbonate, 0.25% (w/v) bovine serum albumin (BSA) (Sigma-Aldrich, A1470), and 0.1 mg/mL kanamycin (Sigma-Aldrich, 11815032). Afterward, the tube was shaken and 14 mL HBSS, supplemented as described above, was added. After centrifugation and resuspension, the digest was filtered over a 500-micron filter (pluriSelect Life Science, 43-50500-50). The pellet was resuspended in 14 mL Histopaque 1100, the latter prepared by mixing Histopaque 1077 (Sigma-Aldrich, 10771) with Histopaque 1119 (Sigma-Aldrich, 11191) and centrifuged for 20 min. at 328 g without brake. The supernatant containing the islets was transferred to a new tube containing 25 mL HBSS, supplemented as described above. After centrifugation, the islets were resuspended in islet culture medium and were allowed to recover at 37°C and 5% CO2 for at least 2 hours before handpicking and subsequent overnight culture. After overnight culture, mouse islets were collected in a 1.5 mL tube, centrifuged (3 min. at 124 g) and dissociated into single cells with NeuroCult Dissociation Solution (StemCell Technologies, 05715). For fluorescence-activated cell sorting (FACS), cells were washed with RPMI 1640 + 10% (v/v) FBS before filtering over a 5 mL filter cap tube (Corning, Falcon, 352235). For scRNA-seq, cells were washed with ice-cold PBS + 0.04% (w/v) BSA before filtering with a 40-micron Flowmi cell strainer (Sigma, BAH136800040) into a new 1.5 mL tube on ice. Cell number and viability were measured with a NucleoCounter NC-200 using a Via1 cassette (ChemoMetec, 941-0012). For scRNA-seq of islet cells of 3-week-old CTRL and β-Tfrc-KO mice, GEMs and scRNA-seq libraries were prepared with the Chromium Next GEM Single Cell 3' Kit v3.1, Chromium Next GEM Chip G Single Cell Kit, and the Dual Index Kit TT Set A according to the manufacturer's instructions using the Chromium Controller (10x Genomics). GEM reverse transcription (GEM-RT) was carried out in a C1000 Touch Thermal Cycler (Bio-Rad) at 53°C for 45 min., followed by inactivation at 85°C for 5 min. Cleanup of the GEM-RT product was performed using Dynabeads MyOne SILANE reagent and the SPRIselect Reagent Kit (Beckman Coulter). cDNA amplification involved an initial denaturation at 98°C for 3 min., followed by 11 to 12 cycles at 98°C for 15 sec., 63°C for 20 sec., and 72°C for 1 min., with a final extension at 72°C for 1 min. Subsequent steps included enzymatic fragmentation, size selection, and the addition of adaptors and sample indices. Sequencing libraries were loaded onto a NovaSeq 6000 using the Novaseq S2 Reagent Kit (Illumina).