RNA-seq of homo sapiens - cornea
mRNA libraries were prepared from 1ug of total RNA using the QIAseq Stranded RNA Library Kits following the manufacturer's instructions. The library was purified using ice-cold ethanol concentrations and were checked using Agilent 4150 Tape Station followed by a qubit 3.0 flourometer. After RNA fragmentation step, the reverse transcription step generates first strand cDNA. Strand specificity is ensured by optimized combinations of RT enzyme and buffer In the second strand synthesis step, a special combination of enzymes with carefully adapted buffer formulations allow the degradation of RNA, the generation of a second cDNA strand and the generation of blunt DNA ends and A-base addition necessary for efficient ligation of the Illumina compatible adapters. The CleanStart PCR mix efficiently amplifies the generated RNA-seq library irrespective of high GC or AT sequences. The paramagnetic QIAseq Beads included with kits provide fast and efficient reaction cleanup.