RNA-seq in EndoCbetaH5 under high glucose, treated or not with naltrindole
In human EndoCßH5 cells, i.e. post-mitotic mature pancreatic beta cells that endogenously express OPRD1 encoding delta opioid receptor (transcripts per million [TPM] of 11 according to our RNA-seq data), a specific antagonist for delta opioid receptor (naltrindole [NTI]) significantly increased insulin secretion in high glucose condition. We performed RNA-seq in these cells to decipher signalling pathways under delta opioid receptor antagonism. Overall design: RNA-seq in EndoCßH5 cells. For this experiment, we used the high glucose condition (16.7 mM glucose) supplemented or not with 10 µM naltrindole (naltrindole hydrochloride, Sigma-Aldrich). RNA was extracted from EndoCßH5 cells using the NucleoSpin RNA kit (Macherey-Nagel, Düren, Germany), following manufacturer's recommendations. 100 ng total RNA was used to perform library preparation using the NextFlex Poly(A) Bead 2.0 (v21.01; PerkinElmer Waltham, USA). Sequencing was done on the NovaSeq6000 system (flowcell SP, Illumina) using a paired-end 2×100 bp protocol. Raw data were demultiplexed using bcl2fastq v2.20.0.422 (Illumina). An adapter trimming step was done using trimmomatic version 0.39 (MINLEN:35 AVGQUAL:20). Mapping of reads on the human genome (Hg38) was performed using STAR (v2.7.3a). On average, 49 million reads accurately mapped against the human genome. Raw and normalized counting steps were done using RSEM (v1.3.1) using a GTF from Encode (version 39), and Ensembl (version 105) for gene name annotations. Differential analysis was performed using DESeq2 (v1.24.0). Gene-set enrichment analysis was done using Metascape (accessed in April 2022)