Qubit rna assay kit in qubit 2.0 fluorometer

RNA degradation and contamination was monitored on 1% agarose gels. RNA purity was checked using the NanoPhotometer^® spectrophotometer (IMPLEN, CA, United States). RNA concentration was measured using Qubit® RNA Assay Kit in Qubit^® 2.0 Fluorometer (Life Technologies, CA, United States). A total amount of 3 μg total RNA per sample was used as input material for the small RNA library. Sequencing libraries were generated using NEBNext^®Multiplex Small RNA Library Prep Set for Illumina^® (NEB, United States.) following manufacturer’s recommendations and index codes were added to attribute sequences to each sample. Briefly, Total RNA was ligated to the RNA 3′ and RNA 5′ adapters and reverse transcription, followed by PCR, was performed to make cDNA constructs of the small RNAs. PCR products were purified on a 8% polyacrylamide gel (100 V, 80 min). DNA fragments corresponding to 140 ~ 160 bp (the length of small non-coding RNA plus the 3′ and 5′ adaptors) were recovered and dissolved in 8 μl elution buffer. At last, library quality was assessed on the Agilent Bioanalyzer 2,100 system using DNA High Sensitivity Chips. We then performed single-end sequencing (50 bp) on an Illumina Hiseq2500 at the LC-BIO (Novogene Experimental Department, Beijing, China) following the vendor’s recommended protocol.

Total RNA of each sample was isolated using TRIzol reagent (Invitrogen, Carlsbad, CA, USA). The NanoPhotometer spectrophotometer (IMPLEN, Westlake Village, CA, USA), the Qubit RNA Assay Kit in Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA), and the RNA Nano 6000 Assay Kit of Bioanalyzer 2100 System (Agilent Technologies, Santa Clara, CA, USA) ware utilized to check the purity, concentration, and integrity of RNA respectively. Eight cDNA libraries in total were constructed from ox-LDL-treated rabbit VSMCs (n = 4) and control group (n = 4). Sequencing libraries were generated using NEB-Next Ultra Directional RNA Library Prep Kit for Illumina (NEB, Ipswich, MA, USA) followed by library fragments purification and quality assessment. Finally libraries were sequenced on Illumina HiSeq 4,000 Platform, and paired-end reads with 150 bp were generated by Illumina HiSeq 2,500 Platform. We screened out clean reads of high-quality from raw data (66.1 GB, SRA accession ID: SRP124805) for subsequent analyses. The rabbit (Oryctolagus cuniculus) genome (OryCun2.0 in the NCBI Assembly database) was used as reference genome for reads mapping through TopHat v2.0.9 (Kim et al., 2013 (link); Trapnell, Pachter & Salzberg, 2009 (link)). Cufflinks v2.1.1 was used for mapped reads assembly of each sample (Trapnell et al., 2010 (link)).

RNA qualification and quantification were determined by electrophoresis analysis at 1% agarose gels to monitor whether its degradation and contamination. RNA purity was measured by utilizing NanoPhotometer® spectrophotometer (IMPLEN, CA, USA). RNA concentration was determined using a Qubit® RNA Assay Kit in Qubit® 2.0 Fluorometer (Life Technologies, CA, USA). RNA integrity was assessed using the RNA Nano 6000 Assay Kit of the Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA).