title: RNA-sequencing of isolated pancreatic acinar cells from RalGAPβ knockout mice against control mice
design: RNA-sequencing of isolated pancreatic acinar cells from RalGAPβ knockout mice against control mice
library_construction_protocol: Pancreata were resected, washed in PBS and minced into small pieces (1-2 mm) in 2 ml RPMI-1640 without phenol red (Sigma-Aldrich, R7509) supplemented with 2 % FBS (Biowest, S1810), 10 mM L-glutamine (Sigma-Aldrich, G7513) and 50 U Penicillin-Streptomycin (Thermo Fisher Scientific, 15070063). After two washing steps with 5 ml RPMI/2% FBS, each minced pancreas was digested in 2 mg/ml Collagenase, Type 4 (Worthington-Biochemical, LS004188), 2 mg/ml Dispase II (Sigma-Aldrich, D4693) and 20 µg/ml DNase I (AppliChem, A3778) dissolved in 15 ml HBSS (Sigma-Aldrich, H6648) at 37 °C with 5 % CO2 for 1 h, including thorough pipetting every 15 min to enhance dissociation. The isolated cells were then washed two times with 35 ml. PBS (400xg, 5 min, 4 °C). The cells were resuspended in 0.5 ml RPMI/2 % FBS, filtered through a 35 µm cell strainer, the filter washed with 0.5 ml RPMI/2 % FBS and 0.1 mg/ml Soybean Trypsin Inhibitor (Thermo Fisher Scientific, 17075029) was added. For acinar cell sorting, the cells were stained 1:25 with rat MPx1 MICO-2A6 (gift from C. Dorrell; Dorrell, C. et al. Isolation of mouse pancreatic alpha, beta, duct and acinar populations with cell surface markers. Mol. Cell. Endocrinol. 339, 144–150 (2011)) for 20 min on ice, then washed three times with 3 ml RPMI/2 % FBS (400xg, 5 min, 4 °C) and stained 1:200 with FITC goat anti-rat (BioLegend, 554016) for 10 min on ice. After a washing step with 3 ml RPMI/2 % FBS (400xg, 5 min, 4 °C), the cells were counterstained with APC rat anti-CD45 (BioLegend, 103112) and PE-Cy7 rat anti-CD19 (BD Biosciences, 55284, clone 1D3) for 20 min. After a final washing step, the cells were stained 1:100 with 7-AAD Viability Staining Solution (BioLegend, 420404). Cell sorting was performed on a FACSAria II using the FACSDiva version 9.0.1 and FACSuite version 1.0.6 software (BD Biosciences). Flow cytometry data were analysed using FlowJo software version 10.5.3 (BD Life Sciences). For RNA library preparation, 0.5-1 x 10^6 MPx1-positive FACS sorted cells were used for RNA isolation using the Quick-RNA Microprep Kit (Zymo Research, R1051) according to manufacturer's instructions. Library preparation of the total RNA was performed with NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (New England Biolabs, E7760) and NEBNext Poly(A) mRNA Magnetic Isolation Module (New England Biolabs, E7490). 