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 <i>Mus
musculus </i>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 <i>Pkd1</i>^Δ/Δ mice <i>Pkd1</i>^Δ/Δ<i>;IL37b/IL37b</i> mice, or control<i> Pkd1</i>^Δ^/+<i> </i>mice and control <i>Pkd1</i>^Δ<i>^/+;IL37b/IL37b </i>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 &lt;0.05. This list was filtered
following count/total sequences log2 transformation for fold changes of ±2.