GSM5964859: HEK293T_C3; Homo sapiens; ncRNA-Seq
Briefly, 1μg total RNA was subjected to alkaline hydrolysis, which was performed in 50 mM bicarbonate buffer pH 9.2 for 12 min at 95℃. The reaction was stopped by ethanol precipitation using 3M NaOAc (Sigma), pH 5.2 and Glycoblue (Invitrogen) as a carrier in liquid nitrogen. The size of generated RNA fragments was assessed by electrophoresis (1% agarose gel, 80V, 50 min) and were ranging from 50 to 200 nt.   RNA fragments were directly 3'-end dephosphorylated using 5 U Antarctic Phosphatase (NEB) for 30 min at 37℃, and then phosphorylated at the 5'-end using T4 PNK and 1mM ATP for 1h at 37℃.Resulting RNA fragments were then purified using Rneasy MinElute Cleanup kit (QIAGEN) according to the manufacturer instructions with minor modification. Finally, 10 μL of nuclease-free water was used to elute RNA fragments. Construction of RNA fragments library was performed by LC Sciences (China) using TruSeq Small RNA Sample Prep Kits (Illumina) following manufacturer instruction. After PCR amplification, products were purified on a 6% poly-acrylamide Tris-borate-EDTA gel. DNA fragments corresponding to 140~270 bp (the length of fragments plus adaptors) were recovered, and were sequenced on Illumina Hiseq2500 platform and single-end 50 bp reads were generated.