biostudies-arrayexpressMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-15030Pancreatic RalGAPβ deficiency induces inflammation and neoplasia. RNA sequencing of isolated acinar cells of cerulein treated mice was performed 24 hours after treatment to identify underlying processes in defective acinar regeneration.biostudies-arrayexpressSample Collection - Pancreata were resected 24 h after the first injection, 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).Nucleic Acid Extraction - 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.Sequencing - Single-end read sequencing was performed using a NextSeq ® 2000 System with a read length of 72 bp. The samples were demultiplexed with the Illumina® DRAGEN™ Bio-IT Platform. Quality control was done using FastQC version 0.11.9. Trimmomatic version 0.38 was used for adapter and low quality end trimming as well as for general quality trimming utilizing a sliding window of 4 bp with a minimal average base quality of 15. Reads below a minimum read length of 15 bp were discarded.Sample Treatment - Acute pancreatitis was induced by administration of 7 hourly intraperitoneal injections of cerulein (MedChemExpress, HY-A0190; 100 ng/g bodyweight in 0.9 % saline) after a fasting period of 12 h (water ad libitum). Control animals received injections with 0.9 % saline.Library Construction - 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).MINSEQE ScoreAssays and DataProcessed DataorganisationMAGE-TAB FilesData Transformation - Gene based read counting was done using HTSeq version 2.0.3 with the Ensembl annotation version 92. Differential expression analysis was performed using the R package DESeq2 version 1.40.1. The R package org.Mm.eg.db version 3.17.0 was used to convert Ensembl IDs to mgi symbols.Sequence Alignment - The resulting reads were aligned to the Ensembl GRCm39 reference genome, using HISAT2 version 2.2.1 and sorted using SAMtools version 1.16.1.UnknownTranscriptomicsGenomicsProteomicsNextSeq 2000RNA-seq of coding RNAMus musculusE-MTAB-15029Andrea OeckinghausLisa ApkenfalseRNA-sequencing of isolated pancreatic acinar cells from RalGAPβ knockout mice against control mice treated with ceruleinPancreatic RalGAPβ deficiency induces inflammation and neoplasia. RNA sequencing of isolated acinar cells of cerulein treated mice was performed 24 hours after treatment to identify underlying processes in defective acinar regeneration.2025-06-03T00:00:00Z2025-06-04T09:38:50.562Z2025-04-15T12:12:37.455ZE-MTAB-15030ERP171648E-MTAB-15029EFO_0002944EFO_0004170EFO_0004917EFO_0005518EFO_0003816EFO_0003738EFO_0003969EFO_0004184