GSM8746995: CD34_W1_; Homo sapiens; RNA-Seq
RNA was isolated using RNAeasy microkit (Qiagen) Libraries were prepared using KAPA Stranded with RiboErase RNA-seq kit (according to the manufacturer's instructions). Briefly, 17–25 ng of starting RNA were subjected first to cytoplasmic and mitochondrial ribosomal RNA (rRNA) depletion by hybridization of complementary DNA oligonucleotides, followed by treatment with RNase H and DNase to remove rRNA duplexed to DNA and original DNA oligonucleotides. Samples depleted of rRNA were then subjected to 94 °C for 6 min in the 2×Fragment, Prime, and Elute Buffer in order to obtain 200–300 bp fragments. cDNA synthesis was run in two steps following the manufacturer's instructions. The ligation step consisted of a final volume of 110 μl of the adaptor ligation reaction mixture with 60 μl of input cDNA, 5μl of diluted adaptor, and 45 μl of ligation mix (50 μl of ligation buffer + 10 μl of DNA ligase). The Kapa Dual- Indexed Adaptors stock concentration was diluted to 1.5mM to get the best adaptor concentration for library construction. The ligation cycle was run according to the manufacturer's instructions. To remove short fragments such as adaptor dimers, 2X AMPure XP bead clean-ups were done (0.63 SPRI and 0.7 SPRI).