GSM8746454: Mettl3 KO-1; Mus musculus; OTHER
RNA extraction was performed by combination of Trizol reagent (ThermoFisher Scientific) and RNeasy mini kit (QIAGEN).100 µg of total RNA from WT or Mettl3KO ROs (day 4) passed QC were deleted less than 200nt of RNA using MEGA clear transcription clean-up kit (Ambion, AM1908), and then underwent PolyA+ selection using Dynabeads Oligo(dT)25 kit (Invitrogen, 61006). After polyA+ selection, checked mRNA purity and quantity by nanodrop, about 800-1,000ng mRNA went through fragmentation using NEB next Magnesium RNA Fragmentation Module (NEB, E6150S). 400-500ng of fragmented RNA with 5% of spike-in probes were added into 40 μl reaction buffer containing 6 μl glyoxal solution (8.8 M in H2O, Sigma-Aldrich, 50649), 20 μl DMSO and nuclease-free water. The mixture was incubated in a preheated thermocycler for 30 min at 50 °C. The tubes were placed at room temperature after incubation, then 10 μl saturated H3BO3 (Sigma-Aldrich, B0394) solution was added and the incubation continued at 50 °C for 30 min. Fifty microliters of deamination buffer was prepared per sample containing 750 mM NaNO2 (Sigma-Aldrich, 31443), 40 mM MES (pH 6.0), 10 μl glyoxal solution (8.8 M in H2O) and nuclease-free water. The 50 μl sample was added to premixed deamination buffer and gently pipetted ten times. The tubes were incubated for 8 h at 16 °C. RNA was purified by ethanol precipitation. The pellets were dissolved by 50 μl deprotection buffer (20ml deprotection buffer combined 10 ml 1 M triethylammonium acetate solution pH 8.6 with 9.5 ml deionized formamide and nuclease-free water to 20 ml). The mixture was incubated at 95 °C for 10 min. RNA was purified by ethanol precipitation and twice clean-up by RNA Clean & Concentrator kit. The RNAs were sent to University of Michigan DNA Sequencing Core for direct RT and library construction, and sequencing using Illumina NovaSeq S4 Libraries platform with PE read lengths of 2 ×150 base pairs.