Name: PHH_D1_2D_RNA_p
Instrument: Illumina HiSeq 2500
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: For primary cultures, cells were obtained from three independent donors and cultured in parallel in both conditions. Cells were grown in sandwich configuration between two layers of extracellular matrix (collagen coated plates and medium containing 0.25mg/ml Matrigel) and harvested at day 9 post seeding. Qiagen RNeasy plus mini kit Total RNA from each sample was used as input for the Illumina TruSeq Stranded messenger RNA LT Sample Prep Kit (Illumina) and sequencing libraries were created according to the manufacturer's protocol. Briefly, poly-A containing mRNA molecules were purified using poly-T oligo-attached magnetic beads. Following purification, the mRNA was fragmented and copied into first strand complementary DNA using random primers and reverse transcriptase. Second strand cDNA synthesis was then done using DNA polymerase I, RNase H and substituting dUTP for dTTP. The cDNA was ligated to adapters and enriched with PCR to create the final cDNA library. The library was pooled and sequenced