title: GSM7468698: FF2_Input; Mus musculus; RIP-Seq
library_construction_protocol: Total RNAs were extracted from langendorf isolated cardiomyocytes using TRIzol Reagent (Invitrogen, cat. NO 15596026）following the methods by Chomczynski et al (DOI:10.1006/abio.1987.9999). DNA digestion was carried out after RNA extraction by DNase I. RNA quality was determined by examining A260/A280 with NanodropTM OneCspectrophotometer (Thermo Fisher Scientific Inc). RNA Integrity (RQN) was confirmed by Qsep 100 (BIOptic Inc). 200 μg total RNAs were used for polyadenylated RNA enrichment by VAHTS mRNA Capture Beads（VAHTS, cat. NO. N401-01/02）. 20 mM ZnCl2 was added to mRNA and incubated at 95℃ for 5min-10min until the RNA fragments were mainly distributed in 100-200 nt. Then 10% RNA fragments was saved as “Input” and the rest was used for m5C immunoprecipitation（IP）.The specific anti-m5C antibody (Abcam, ab10805) was applied for m5C immunoprecipitation. RNA samples of both input and IP were prepared using TRIzol reagent(Invitrogen, cat. NO 15596026). The stranded RNA sequencing library was constructed by KC-DigitalTM Stranded mRNA Library Prep Kit for Illumina® (Catalog NO. DR08502, Wuhan Seqhealth Co., Ltd. China) following the manufacturer's instruction. The kit eliminates duplication bias in PCR and sequencing steps, by using unique molecular identifier (UMI) of 8 random bases to label the pre-amplified cDNA molecules. The library products corresponding to 200-500 bps were enriched, quantified and fnally sequenced on a MGISEQ-T7 instrument (MGI) in PE150 mode.