Illumina HiSeq 4000 paired end sequencing; scRNA-seq of pancreatic islets from control and Haster-pKO mice
scRNA-seq of pancreatic islets from control and Haster-pKO mice
To isolate pancreatic islets, the pancreas was inflated with an ice-cold collagenase P solution (1 mg/mL in Hanks' balanced salt solution (HBSS) buffer, Roche) injected through the main pancreatic duct. The pancreas was then dissected and incubated at 37ºC for 8 min with continuous agitation. The pancreatic tissue was disaggregated by gentle suction through a needle, and cell suspension was washed 4 times with cold HBSS-0.5% BSA. The islet pellet was resuspended in 7 mL Histopaque containing a 7:3 ratio of pre-cooled Histopaque 1077 (Merck) and Histopaque 1119 (Merck), and 7 mL of HBSS-0.5% BSA was layered on top of the Histopaque. The gradient was centrifuged at 950 g for 20 mins at RT. The interphase containing the islets was collected, washed 3 times with HBSS-0.5% BSA and islets were finally further enriched by gentle aspiration with a pipette under a stereomicroscope. Islets were allowed to recover for 2 days in culture in RPMI containing 11 mM glucose supplemented with 10% fetal calf serum and penicillin-streptomycin (1:100; Invitrogen) at 37°C and 5% CO2. Cultured mouse islets were dissociated with Accutase (Merk) for 15 min at 37ºC. Islet cell suspensions were centrifuged at 600g 3 min and resuspended in cell culture medium with DAPI before FACS sorting to remove dead cells and doublets. After sorting, cells were centrifuged at 600g for 3 min and resuspended in PBS/0.04% BSA. Nucleic acid were extracted following the manufacturer's instructions for 10x Genomics Chromium Single Cell 3′ Reagent Kit v3 Libraries were prepared following the manufacturer's instructions for 10x Genomics Chromium Single Cell 3′ Reagent Kit v3