GSM6751706
hiPSC islets were washed with Versene (Thermo Fisher Scientific, 15040066) and incubated with Accutase (Sigma-Aldrich, A6964-100ML) for 10 min at 37°C. Dissociation was stopped by the addition of PBS supplemented with 1% Bovine Serum Albumin (BSA). Dissociated cells were washed twice in PBS (1%BSA) buffer, centrifuged at 300 rcf for 5 min, filtered using Flowmi 40µm tip strainer (Bel-Art, H13680-0040), counted and adjusted to 1  × 10^6 cells ml–1 cells in PBS (1%BSA) for encapsulation. Cell count and viability were measured using the Luna-Fl automated Fluorescence Cell Counter (Logos Biosystems). For each siRNA treatment, 6000 cells were loaded onto the 10X Chromium Single Cell Platform (10X Genomics) using the Next GEM Single Cell 3' library and Gel Bead Kit (v3.1 chemistry) according to manufacturer's instructions (10x User Guide CG000204, Revision D). RNA quality was assessed using Tapestation (Agilent). Libraries were quantified using Tapestation (Agilent), the Qubit 2.0 (ThermoFisher Scientific) and KAPA Library Quantification Kit for Illumina Platform (KAPA Biosystems) before pooling. Libraries were pooled in equimolar amounts for paired-end sequencing on an Illumina NextSeq 2000 instrument to yield ∼168 million (range 147–195 million) 100-bp-long reads on average per sample. 10X Genomics scRNAseq