GSM5288660: HepG2_WT_30min-EU-RNA_rep1; Homo sapiens; OTHER
Then after 10-min or 20-min or 30-min, wash the cells with cold DPBS once and add TRIzol reagent immediately to lyse the cells. Cellular total RNA was extracted by the standard protocol of TRIzol reagent. Then EU-labeled RNA was enriched and purified by Click-iT™ Nascent RNA Capture Kit, for gene expression analysis. THe EU-labeled RNA was fragmented into 40-70 nt using RNA Fragmentation Reagent (AM8740, Invitrogen) following the manufacturer's protocol and Oligo Clean & Concentrator (Zymo Research). Then the library construction procedures will be conducted with T4 PNK end repair, RNA 3'-adapter ligation, HIV RT reverse transcription, cDNA 3'-adapter ligation and PCR amplification respectively, according to standard steps in section “DAMM-seq for methylated site detection”. All libraries were sequenced on Illumina NovaSeq 6000 with single-end 100 bp read length.