Chapter 3: Early Onset RNA-seq DE genes

Snap frozen P4 kidney tissues were crushed using micropestles in RLT Buffer and RNA was extracted from these tissues using QiaShredders followed by Qiagen RNAeasy Mini Kits according to manufacturer’s protocol. RNA concentration and integrity were determined by Aligent bioanalyzer. cDNA library preparation was carried out using NEB Next Ultra Directional RNA Library Prep Kit for Illumina RNA sequencing (E7420S) and sequenced on an Illumina HiSeqX10 machine in 150 bp paired end format. Sequence data was processed with Skewer adaptor trimmer and mapped with HiSat2 to the Mus musculus GRCm38_v90 genome assembly. The resulting ordered Bam files were analysed in Seqmonk v1.43 software using default settings. Samples were grouped as Pkd1 KO (Pkd1^Δ/Δ mice), Aurka KO (Aurka^Δ/Δ mice), Pkd1 double KO (Pkd1^Δ/Δ; Aurka^Δ/Δ mice) or Control Cre (Hoxb7-cre mice). A minimum of 13 million reads was obtained per sample 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.