GSM5904721: Media stimulated cDCs donor 3; Homo sapiens; RNA-Seq
Total RNA was isolated from activated cDCs using the Qiagen RNeasy Mini Kit (Qiagen) or Arcturus PicoPure (Life Technologies) kits following manufacturer's protocols. During RNA isolation, DNase treatment was additionally performed using the RNase-free DNase set (Qiagen). RNA quality was checked using an Agilent 2100 Expert bioanalyzer (Agilent Technologies). RNA quality was reported as a score from 1 to 10, samples falling below threshold of 8.0 were omitted from the study. RNA-seq libraries were prepared with KAPA mRNA HyperPrep kit (Roche) according to manufacturer's instruction. Poly(A) mRNA was isolated from 60 ng total RNA using oligo(dT) magnetic beads and then fragmented at 85°C for 6 minutes, targeting 250-300 bp-range fragments. Fragmented RNA was reverse transcribed by incubating it for 10 minutes at 25°C followed by 15 minutes at 42°C and finally inactivated for 15 minutes at 70°C. This was followed by second strand synthesis at 16°C for 60 minutes. cDNA was purified using AMPure XP beads (Beckman-Coulter), then A-tailed and ligated with Illumina unique adapters (Illumina). Adapter-ligated cDNA was purified using AMPure XP beads and amplified by 10 cycles of PCR. The final libraries were cleaned up using AMPure XP beads and quantified using real-time qPCR (Thermo Fisher). The libraries were sequenced on Illumina HiSeq4000 platform generating paired end reads of 75bp.