GSM4859518: mGro_Ctrl_2; Homo sapiens; OTHER
TRIzol (Invitrogen, 10296010) was added to terminate the reaction and extract RNA, followed by DNA depletion by DNase I digestion. RNA was resuspend in nuclease-free water and adjusted to 200~300 ng/μL. rRNA was depleted by NEBNext® rRNA Depletion Kit (NEB, E0350), and RNA was resuspended in 30 μL nuclease-free water. The efficiency of rRNA depletion was measured by RT-qPCR using PrimeScript™ 1st Strand cDNA Synthesis Kit (TOYOBO, 6110A) and iTaq™ Universal SYBR® Green Supermix (Bio-Red, 1725124). A sequence library was constructed using NEBNext® Ultra II Directional RNA Library Prep Kit (NEB, E7760), with several modifications. Briefly, RNA was resuspended in 5 μL nuclease-free water, and fragmented with 4 μL NEBNext First Strand Synthesis Reaction Buffer and 1 μL Random Primers for 7 min at 94 °C. RT was performed by adding 8 μL NEBNext Strand Specificity Reagent, 5 μL 5× reaction buffer for RT, and 2 μL recombinant HIV-1 reverse transcriptase (Worthington Biochemical Corporation), for 10 min at 25°C, 1 h at 37°C, and then 15 min at 70°C, and finally held at 4°C. Then the following steps followed the manufacture's protocol. The library sizes were validated using Agilent 2100 Bioanalyzer and sequenced on Illumina Hiseq X10 with paired-end 2×150 bp read length.