GSM7814644: HOM_S2, scRNAseq; Mus musculus; RNA-Seq
Following decapitation, pancreata were inflated in situ with freshly prepared collagenase (~ 5 mL of a 0.65 mg/mL solution) that was injected into the bile duct under a dissecting microscope (Supplemental Figure 9). Each inflated pancreas was dissected, placed in a 50 mL centrifuge tube, and then incubated in a 37°C water bath for 15 min. Four digested pancreases of each genotype were pooled together, after which islets were isolated by Histopaque density gradient (Sigma-Aldrich, Cat# 10771 and 11191) centrifugation (1200 rpm, 4°C for 30 min). All detectable islets were handpicked under a microscope (at 10x magnification) and placed into a 15 mL centrifuge tube containing 1 mL of 1x HBSS media (GibcoTM, ThermoFisher Scientific, Waltham, MA; Cat# 14025-076). Single cell dispersion was then carried out as follows: islets were centrifuged (200 rpm, 4°C for 3 min), supernatant was removed, islets were incubated with 2 mL Accutase® solution (Innovative Cell Technologies, Inc. San Diego, CA; Cat# AT104) in a 37°C water bath for 15 min, after which cells were dispersed by pipetting up and down ten times using a 1 mL micropipette every 5 min x 3. The digestion was stopped by adding 10 mL of prewarmed RPMI-1640 (GibcoTM, ThermoFisher Scientific, Waltham, MA; Cat# 11875-085) containing 10% FBS (Atlanta Biologicals, Flowery Branch, GA; Cat# S11150). The cell solution was filtered through a 40 μm cell strainer and centrifuged (200 rpm at 4°C for 3 min), after which the cells were resuspended with 2 mL of fresh media. A total of three washes were performed before the final resuspension of islet cells in 0.5 mL of serum containing media. The cells were placed on ice and then submitted for fluorescence-activated cell sorting (FACS) within 20 minutes. cDNA and single-cell libraries were prepared with the Chromium Next GEM Single Cell 3ʹ Reagent Kit v3.1 (Dual Index) (10x Genomics, Pleasanton, CA), as per manufacturer's instructions: Cells in suspension were first prepared as gel beads in emulsion (GEMs) on a Single Cell 3' Chip G (10x Chromium) using the Chromium Controller (10x Genomics). For this, ~1,600-1,800/µL cells were loaded, aiming for 10,000 cells per channel. Barcoded RNA transcripts in each single cell were reverse transcribed within GEM droplets. cDNA in GEM reaction mixture was purified with DynaBeads MyOneTM Silane beads (ThermoFisher Scientific, Waltham, MA). Read 1 primer sequence was added and full-length barcoded cDNA was amplified for subsequent library construction. cDNA quality was verified by Agilent Tapestation 4200 (Agilent Technologies, Santa Clara, CA) using DNAHS 5000 tape, and concentration was determined with a Qubit 4 Fluorometer (ThermoFisher Scientific) using the DNA HS assay before sequencing. Sequencing libraries were prepared by fragmentation, end-repair, ligation with indexed adapters, and PCR amplification using the Chromium Single Cell 3' library kit v3.1 (10x Genomics). Nucleic acid was cleaned up using Ampure XP beads (Beckman Coulter Inc, Brea, CA). Post library preparation quality control was performed using DNA 1000 tape on the Agilent Tapestation 4200 and quantified by Qubit and real-time quantitative PCR on a LightCycler 96 System (Roche, Basel, Switzerland). Pooled libraries were sequenced at 1.6 pM on the Illumina NextSeq 2000 P2 100 Flowcell at a configuration of 28 x 90 x 10 x 10 base-pair, which codes for 28 base-pair Cell Barcodes, 90 base-pair Read, 10 base-pair unique molecular identifiers (UMI) and 10 base-pair Sample index. Cell Ranger Single-Cell Software Suite (v 5.0.1, 10X Genomics) was used to demultiplex cellular barcodes, map reads to the genome and transcriptome using the STAR aligner, and down-sample reads as required to generate normalized data across samples