Illumina NovaSeq 6000 paired end sequencing; Gene expression profiling of Ercc1-/- and age-matched wt pancreata
Gene expression profiling of Ercc1-/- and age-matched wt pancreata
Total RNA was isolated from wt and Ercc1-/- pancreata  Total RNA isolation kit (Qiagen) as described by the manufacturer For RNA-Seq, libraries were prepared using the Illumina® TruSeq® mRNA stranded sample preparation kit. Library preparation started with 1μg total RNA. After poly-A selection (using poly-T oligo-attached magnetic beads), mRNA was purified and fragmented using divalent cations under elevated temperature. The RNA fragments underwent reverse transcription using random primers. This is followed by second strand cDNA synthesis with DNA Polymerase I and RNase H. After end repair and A-tailing, indexing adapters were ligated. The products were then purified and amplified (14 PCR cycles) to create the final cDNA libraries. After library validation and quantification (Agilent 2100 Bioanalyzer), equimolar amounts of library were pooled. The pool was quantified by using the Peqlab KAPA Library Quantification Kit and the Applied Biosystems 7900HT Sequence Detection System.