GSM1423263: RAFLS from RA before therapy, untreated replicate 2; Rattus norvegicus; RNA-Seq
Once  in vitro cultures ended, cells were harvested enzimatically with trypsin (Pierce) treatment, collected by centrifuged and resuspended in RNA Later before  -80ºC storage. Total RNA was then extracted and purified using Trizol Reagent (Invitrogen) according to the manufacturer's instructions, checked for the RIN number to inspect RNA integrity by the Agilent Bioanalyzer 2100 following the manufacturer's procedures of RNA 6000. Nano Kit  (Cat#5067-1511, Agilent Technologies). The total RNA samples were first treated with Dnase I to degrade any possible DNA contamination. Then the mRNA was enriched by using the oligo(dT) magnetic beads. Mixed with the fragmentation buffer, the mRNA was fragmented into short fragments (~200bp). Then the first strand of cDNA was sythesized by using random hexamer-primer. Buffer, dNTPs, RNase H and DNA polymerase I were added to synthesize the second strand. The double strand cDNA was purified with magnetic beads. End reparation and 3'-end single nucleotide A addtion was then performed. Finally sequencing adaptors were ligated to the fragments. The fragments were enriched by PCR amplification. During the QC step, Agilent 2100 Bioanalyzer and ABI StepOnePlus Real-Time PCR system were used to qualify and quantify the sample library.