Gotaq 2 step rt qpcr dye based detection system

RNA was extracted from cells using the miRNeasy miRNA isolation mini kit (#217004, Qiagen). To quantify mRNA of interest, cDNA synthesis and quantitative PCR (qPCR) were conducted using the GoTaq 2-Step RT-qPCR dye-based detection system (#A6010, Promega) and a Roche 480 Lifecycler (Roche, Sussex, UK). Quantification of microRNAs was achieved using the MystiCq microRNA cDNA synthesis mix and MystiCq microRNA qPCR dye-based assay primer system (#MIRRT, Sigma). Primers are detailed in Supplementary Table 3. For RNAseq samples were prepared using the Truseq small RNA library (#RS-200-0012, Illumina, Essex, UK), Truseq Stranded RNA library (#20020597, Illumina) and sequenced using the NextSeq 500 High Output Run (150 cycles) for two biological replicates. Two biological replicates were sequenced and analysis conducted using Cutadtapt, SHRiMP, SAMtools for trimming, mapping to GRch38 build, generating the raw counts. Data available from the Gene Expression Omnibus GSE117744. FunRich software was used to conduct enrichment analysis with Fishers exact test to generate P values [45 (link)].