INV7
Prior to library preparation the RNA quality and integrity was assessed according to Illumina guidelines. Library preparation was done using the TruSeq® Stranded mRNA sample preparation 96 rxn kit (Illumina™) following the low sample protocol according to manufacturer’s recommendations. Briefly, approximately 2.5 µg of total RNA was diluted and purified using RNA purification beads targeting the poly-A tail of the mRNA and subsequently was fragmented by means of the enzymes provided in the kit. After the cDNA synthesis adenylation of 3’ ends and ligation of the adaptors were employed. Adaptors were ligated in 12-plex formations, allowing the pooling of 12 samples after the PCR enrichment of the library. Subsequently, the library was quantified using PicoGreen® dye (Life Technologies™) as described in the manufacturer’s protocol. Thereafter 12 samples were pooled at equal concentrations to create the eight pools. In order to accurately quantify the concentration in nM of our pools, the Kapa SYBR® FAST universal qPCR kit (Kapa Biosystems™) for Illunima™ sequencing was used to quantify the number of the amplifiable molecules in the pools and the Bioanalyzer® (Agilent Technologies™) to determine the average fragment size of the pools. These measurements allowed optimizing the flow cell clustering and proceed with the Run. The samples were sequenced in five lanes of an Illumina HiSeq2000 sequencer, for 50 bp pair-end read.