Test, exposed to uranium 30 min
The RNA-Seq PE sequencing library preparation of QC (Quantity/Quality check) passed samples were carried out using illumina TruSeq Stranded m-RNA sample preparation Kit. Total RNA (5 g) samples were processed to isolate poly(A)-tailed mRNA using poly-T attached magnetic beads, followed by enzymatic fragmentation and 1st strand cDNA synthesis (using SuperScript II and Act-D mix). The 1st strand cDNA was used as a template to synthesize second strand using second strand mix. The obtained double strand cDNA (dscDNA) was purified using AMPure XP beads followed by A-tailing, adapter ligation and enrichment by PCR cycles (25 cycles). Libraries were checked with the Agilent 4200 Tape station system (Agilent technologies) using high sensitivity D1000 screen tape as per manufactures protocol and quantified using Qubit fluorometer. For all samples, the mean library fragment size ranged between 434 to 465 bp. Equimolar quantities of Y. lipolytica cDNA libraries were pooled together and subjected to cluster generation and sequencing on 2 X 75 bp PE Illumina NextSeq500 platform. Paired end sequencing was used to enable template fragments to be sequenced in both the forward and reverse directions.