GSM7770086: 293T, con1; Homo sapiens; OTHER
total RNA was extracted by Trizol in accordance with the manufacturer's protocol. mRNA were purified by VAHTS mRNA capture beads (Vazyme). 300 ng mRNA were applied GLORI step.In brief, RNA was fragmented by incubation at 70 °C for 5 min with Zinc Fragmentation Buffer. RNA was added into 40 μl reaction buffer containing 6 μl glyoxal solution (Sigma-Aldrich, 50649), 20 μl DMSO and nuclease-free water. The mixture was incubated for 30 min at 50 °C. Then 10 μl saturated H3BO3 (Sigma-Aldrich, B0394) solution was added and the incubation continued at 50 °C for 30 min following cooled on ice for next use. The 50-μl sample was added to premixed deamination buffer and gently mixed. The tubes were incubated for 8 h at 16 °C. RNA was purified by Dynabeads™ MyOne™ Silane (Thermo Fisher Scientific, cat. no. 37002D). The silane beads were dissolved by 50 μl deprotection buffer as before(文献XXX). The mixture was incubated at 95 °C for 10 min. RNA was purified by silane beads. RNA samples were then treated by PNK (NEB, M0201S) to achieve dephosphorylation of 3′ ends. The RNA was purified by silane beads and then 3′ adapter (sequence:/5rApp/AGATCGGAAGAGCGTCGTG/3Bio/) ligation was performed using T4 RNA ligase2, truncated KQ (NEB, M0373S). The excess adapter was digested by 5′ deadenylase (NEB, M0331S) and RecJf (NEB, M024S). The RNA was purified by silane beads and then subjected to reverse transcription by SuperScript™ III (Thermo Fisher Scientific, cat. no.18080051). The cDNA was purified by silane beads and then cDNA adapter (sequence:    /5Phos/NNNNNNAGATCGGAAGAGCACACGTCTG/3SpC3/) ligation was performed using T4 RNA Ligase 1 (ssRNA Ligase), High Concentration (New England BioLabs, cat. no. M0437M). Library were constructed by PCR with index primers. Libraries were constructed using VAHTS Universal V8 RNA-seq Library Prep Kit for Illumina (Vazyme)