GSM7932280: ko_ctrl2; Mus musculus; RNA-Seq
Total RNA was collected using Qiagen RNeasy Mini kit RLT buffer (Qiagen, 74104) supplemented with 1% 𝛽-mercaptoethanol (BME).  Cells were lysed by passing through a 22-gauge needle, and RNA was isolated following the manufacturer's instruction with RPE buffer supplemented with 1% BME. 10 µg total RNA was treated with 2,2,2-trifluoroethylamine (TFEE) and sodium periodate (NaIO4) at 45 °C for 1 h to convert 4sU to N4-trifluoroethylcytosine.  RNA was then purified twice using RNAClean beads (Beckman Coulter, A63987). 1 µg of treated RNA was used for RNA-seq library preparation using NEBNext®rRNA Depletion Kit(NEB, E6310X) and NEBNext®Ultra™II Directional RNA Library Prep Kit (NEB, E7760L).  Library quality was assessed on Agilent TapeStation4200 using High Sensitivity DNA ScreenTape (Agilent, 5067-5584).  Library was quantified on Qubit4 Fluorometer using Qubit 1X dsDNA HS Assay Kit (ThermoFisher, Q33231).