GSM9032386: pancreatic islet; Mus musculus; RNA-Seq
Islets were isolated from the pancreas of 3 Ide-/- and 3 Ide+/+ C57BL/6 mice. Ice-cold collagenase P (Roche; 0.76 mg/ml in HBSS) was injected through a catheter introduced into the pancreatic duct (2 mL/mouse). The instilled pancreas was excised and digested for 10 min at 37 °C, digestion was stopped by addition of cold HBSS containing 10% heat inactivated FCS. The islets were handpicked under the microscope and dissociated into single cell suspensions by incubation in 100 mL of Accutase (Thermofisher) at 37°C and 5% CO2 for 15 min. The reaction was blocked with 1mL HBSS/10% FCS and the cells were filtered through a 40 mm cell strainer (Corning) and centrifuged at 300 x g for 5 min. The cells were resuspended in 100 mL of cold Sample Buffer (BD Rhapsody) and stained with the viability markers, Calcein AM (Thermo Fisher Scientific) and Draq7 (BD Bioscience) according to the BD Rhapsody Single Cell Analysis protocol. The cell concentration and viability were determined with BD Rhapsody Scanner. Cell barcoding was performed using Custom BD Single Cell Sample Multiplexing Kit (N°626545) after pooling the same number of cells for each genotype. Cell capture and cDNA library amplification were carried out using the BD Rhapsody Enhanced (N°66487), cartridge Reagent Kit (No. 633733) and the BD Rhapsody cDNA Kit (N°633773), respectively, according to the manufacturer's instructions. The DNA library, from 14755 single cells captured in a single run with 4 pooled barcoded samples, was constructed using the BD Rhapsody Whole Transcriptome Analysis (WTA) Pipeline version 2 (N°633801). The Illumina primer R1 was added to the library. The single cell library was quantified by Qubit 4 Fluorometer (Thermofischer), and by capillary electrophoresis using Fragment Analyzer (Agilent Technologies). The sequencing was performed on a NovaSeq 6000 system (Illumina) with 3% control PhiX library (Illumina) on a Flow Cell NovaSeq S1 100cycles to generate ~1500 millions paired-end reads (51 bases + 71 bases).  The generated FASTQ files were uploaded to the SevenBridges platform using the command-line interface (CLI) provided by BD.