GSM5733965: KO_3; Homo sapiens; OTHER
Total RNA was extracted using Trizol and isopropanol (Honeywell, 33539) precipitation, followed by DNAse I Turbo (Invitrogen) treatment for DNA removal. Then, further acidic phenol (Ambion) purification and ethanol precipitation was performed. Briefly, small RNAs (<200 bp) were selected using mirVana miRNA isolation kit (Invitrogen) following the manufacturer's instructions. tRNA libraries were generated using TruSeq Small RNA Preparation Kit (Illumina). Briefly, 3'adenylated and 5'phosphorylated adapters suitable for Illumina RNA sequencing were ligated to the small RNA fraction. RNA was reverse-transcribed at 50ºC for 1 h (SuperScript III cDNA synthesis kit, Invitrogen), followed by PCR amplification with Phusion DNA polymerase (Thermo scientific) and size-selected before sequencing. All samples were multiplexed and sequenced in HiSeq platform (Illumina). Empty vector clone 4 was used as control cell line, and clone 1 as METTL1-KO cell line. Borohydride RNA Sequencing (Bo-Seq)