GSM5866903: HPNE_Ctrl; Homo sapiens; RNA-Seq
Total RNA was isolated using TRIzol (Invitrogen) according to the manufacturer's instructions. Illumina TruSeq RNA Sample Prep Kit (Cat#FC-122-1001) was used with 1 ug of total RNA for the construction of sequencing libraries. RNA library for lncRNA-seq was prepared as rRNA depletion and stranded method. Briefly, the ribosomal RNA was depleted from total RNA using the rRNA Removal Kit following manufacturer's instruction. RNA was then fragmented into 250~300 bp fragments, and first strand cDNA was reverse transcribed using fragmented RNA and dNTPs. RNA was degraded using RNase H, and second strand cDNA was synthesized using DNA polymerase I and dNTPs. Remaining overhangs of double-strand cDNA were converted into blunt ends via exonuclease/ polymerase activities. After adenylation of 3' ends of DNA fragments, sequencing adaptor were ligated to the cDNA. In order to select cDNA fragments of preferentially 250~300 bp in length, the library fragments were purified with AMPure XP system. Uridine digestion was performed using Uracil-N-Glycosylase, which was followed by the cDNA amplification using PCR.