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 Mus musculus 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 Pkd1 KO (Pkd1^Δ/Δ mice), Pkd1 double KO (Pkd1^Δ/Δ;Aurka^Δ/Δ mice) or control (Pkd1^f/f;Aurka^f/f 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<0.05 and independent filtering applied. This list was filtered following count/total sequences log2 transformation for fold changes of ±1.5.