GSM4785312: C_2; Homo sapiens; RNA-Seq
Total RNA was isolated from cultured cell lines. Beads containing oligo (dT) were used to isolate poly(A) mRNA from total RNA. Purified mRNA was then fragmented in fragmentation buffer. Using these short fragments as templates, random hexamer-primers ere used to synthesize the first-strand cDNA. The second-strand cDNA was synthesized using buffer, dNTPs, RNase H and DNA polymerase I. Short double-stranded cDNA fragments were purified with a QIAquick PCR extraction kit (vendor) and eluted with EB buffer for end repair and the addition of an 'A' base. Next, the short fragments were ligated to Illumina sequencing adaptors. DNA fragments of a selected size were gel-purified and amplified by PCR. The amplified library was sequenced using Illumina HiSeq 2000. RNA libraries were prepared for sequencing using standard Illumina protocols