Chapter 5: IL37b RNA-seq DE genes

Snap frozen P0 kidneys were crushed using micropestles in RLT Buffer and RNA was extracted using QiaShredders followed by Qiagen RNAeasy Mini Kits according to manufacturer’s protocol. RNA was sent to GeneWiz for quality assessment, library preparation and sequencing, using the Illumina HiSeqX10 sequencer. Sequence data was uploaded onto Galaxy servers and processed with an automatic adapter trimmer and mapped with HiSat2 to the Mus musculus GRCm38_v100 genome assembly. The resulting Bam files were exported and analysed in Seqmonk v1.47.1 software using default settings. Samples were grouped as Pkd1Δ/Δ mice Pkd1Δ/Δ;IL37b/IL37b mice, or control Pkd1Δ/+ mice and control Pkd1Δ/+;IL37b/IL37b mice. A minimum of 23 million reads was obtained per samples and library duplication and QC metrics were assessed. Differentially expressed genes between groups were determined using the count based DeSeq2 method with a multiple testing corrected p-value cut off of <0.05. This list was filtered following count/total sequences log2 transformation for fold changes of ±2.