GSM5993683: PC3_RCAN3_negative control plasmid; Homo sapiens; RNA-Seq
Total RNAs were extracted from samples 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 DNaseI. RNA quality was determined by examining A260/A280 with NanodropTM OneCspectrophotometer (Thermo Fisher Scientific Inc). RNA Integrity was confirmed by 1.5% agarose gel electrophoresis. Qualified RNAs were finally quantified by Qubit3.0 with QubitTM RNA Broad Range Assay kit (Life Technologies，Q10210). 2 μg total RNAs were used for stranded RNA sequencing library preparation using Ribo-off rRNA Depletion Kit (Human/Mouse/Rat) (Catalog NO. MRZG12324, Illumina) and 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.