GSM5625392: input.filtlowGC; Homo sapiens; RIP-Seq
RIP assay was conducted with antibodies against Ago2 or IgG according to the protocols provided in the Magna RIP™ RNA-Binding Protein Immunoprecipitation kit (Millipore). RNAs were extracted from the immunoprecipitation product, and RT-qPCR was utilized to quantitatively determine the level of HOXC-AS3 pulled down by Ago2 or IgG. The RNA of input and IP was extracted using TRIzol reagent(Invitrogen, Invitrogen, cat. NO 15596026). The stranded RNA sequencing library was constructed by using 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 finally sequenced on Novaseq 6000 sequencer (Illumina) with PE150 model.