GSM8353844: HS980_notrans_GEX; Homo sapiens; RNA-Seq
The SC-islets were rinsed twice in D-PBS without Ca2+ and Mg2+ and then incubated with TrypLE for 15-20 minutes on an orbit shaker at 37°C. The SC-islets were dissociated into single cells by pipetting 15 times using a P1000 pipette, centrifuged at 300 g for 5 minutes, resuspended into D-PBS without Ca2+ and Mg2+ containing 0.04% BSA (Sigma; A7284), and then filtered using a 40 μm cell strainer (VWR; 732-2760).  Mouse iris transplanted with 300 SC-islets was excised at 20 weeks post-transplantation. Excess iris tissue surrounding SC-islet grafts was removed with a scalpel under a stereomicroscope. The remaining iris tissue with SC-islet grafts was washed twice with 2.5 ml D-PBS without Ca2+ and Mg2+ and then incubated in 2.5 ml dissociation buffer (D-PBS without Ca2+ and Mg2+) containing 2 mg/ml Collagenase IV (ThermoFisher; 17104019), 10 U/ml DNase I (Merck; 04716728001), and 1 mg/ml Papain (Sigma; 76216) for 50 minutes in ULA 6-well plate on an orbit shaker at 37°C. The iris tissue was dissociated into single cells by pipetting 15 times using P1000 pipette every 10 minutes. At the end of dissociation, 2.5 ml D-PBS without Ca2+ and Mg2+ containing 10% FBS were added to block the enzymes. Cells were filtered through 40 μm cell strainer, pelleted at 300g for 5 minutes, and resuspended into 0.8 x 106 cells/ml in D-PBS without Ca2+ and Mg2+ containing 1% BSA. The 10x Genomics Chromium Next GEM Single Cell 3ʹ Reagent Kits v3.1 (10x genomics, CG000315 or CG000388) was used for construction of scRNA-seq libraries, sometimes together with Cell Multiplexing Oligo Labeling protocol (10x Genomics, CG000391), followed by RNA sequencing on Illumina Nextseq 2000 machine.