title: RNAseq of human: nucleus pulposus cells
design: Total RNA was isolated using RNeasy mini kit (Qiagen, Germany). Paired-end libraries were synthesized by using the TruSeq RNA Sample Preparation Kit (Illumina, USA) following TruSeq RNA Sample Preparation Guide. Briefly, The poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. Following purification, the mRNA is fragmented into small pieces using divalent cations under 94 for 8 min. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers. This is followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. These cDNA fragments then go through an end repair process, the addition of a single A base, and then ligation of the adapters. The products are then purified and enriched with PCR to create the final cDNA library.