Rnase free te buffer

Validation of differentially expressed genes identified via RNA-seq was carried out by qRT-PCR using KAPA SYBR FAST Universal qRT-PCR master mix (KAPA Biosystems, Woburn, MA, USA) and M-MLV Reverse Transcriptase (Promega, Madison, WI, USA). Total RNA was extracted as above and was quantified using a Nanodrop 2000 (Thermo Scientific, Waltham, MA, USA). Samples for comparison were normalized to a concentration of 50 ng μl⁻¹ using RNAse free TE buffer (Ambion). qRT-PCR analysis was performed using a two-step reaction; cDNA synthesis (37 °C for 15 min and 95 °C for 10 min) followed by qRT-PCR (40 cycles of 95 °C for 10 s, 55 °C for 30 s). Individual reactions were performed in triplicate to eliminate technical variance and reaction for each gene to be analysed was performed in biological triplicate. qRT-PCR reactions were carried out using the ECO Real-Time PCR System (Illumina) according to the manufacturers specifications and the data were analysed according to the 2^−ΔΔCt method (Livak and Schmittgen, 2001 (link)) using the housekeeping gene gapA as an internal control. All primers used are listed in Supplementary Table S4.