GSM3553655: cDC rep3; Mus musculus; RNA-Seq
RNeasy Micro kit (Qiagen) RNA-Seq libraries were prepared with KAPA Stranded mRNA-Seq kit (Roche) according to manufacturer's instruction. First, poly A RNA was isolated from 100ng total RNA using oligo-dT magnetic beads. Purified RNA was then fragmented at 85C for 6 min., targeting fragments range 250-300bp. Fragmented RNA is reverse transcribed with an incubation of 25C for 10mins, 42C for 15mins and an inactivation step at 70C for 15mins. This was followed by second strand synthesis at 16C, 60mins. Double stranded cDNA fragments were purified using Ampure XP beads (Beckman). The dscDNA were then A-tailed, and ligated with illumina adaptors. Adaptor-ligated DNA was purified using Ampure XP beads. This is followed by 10 cycles of PCR amplification. The final library was cleaned up using AMpure XP beads. Sequencing was performed on Illumina NextSeq platform generating paired end reads of 76bp.