Chapter 3: Adult Onset RNA-seq DE genes

Snap frozen 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_v10 genome assembly. The resulting
ordered Bam files were exported and analysed in Seqmonk v1.43 software
using default settings. Samples were grouped as <i>Pkd1</i> KO (<i>Pkd1</i>^Δ/Δ mice), <i>Pkd1</i> double KO (<i>Pkd1</i>^Δ/Δ;<i>Aurka</i>^Δ/Δ mice) or control (<i>Pkd1</i><i>^f/f</i><i>;Aurka</i><i>^f/f</i> mice). A minimum of 24 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
p&lt;0.05 and independent filtering applied. This list was filtered following
count/total sequences log2 transformation for fold changes of ±1.5.