GSM2546872: SMC-IL rep1; Homo sapiens; RNA-Seq
total RNAs were isolated from SMCs or ECs using TRIzol reagents (Invitrogen) followed by removal of ribosomal RNA using the RiboMinus Kit (Invitrogen). Confirming high quality of purified mRNAs, the A260/A280 ratio was determined to be > 1.9 by NanoDrop (Thermo Scientific), and the RNA integrity number was measured to be > 7 using the BioAnalyzer RNA 6000 Pico Kit (Agilent). The cDNA libraries were prepared using Ovation RNA-seq System v2 (NuGEN), and unique barcode sequences were incorporated in the adaptors for multiplexed high-throughput sequencing. The size distribution and concentration of the final product were assessed using the BioAnalyzer High Sensitivity DNA Kit (Agilent) and the Kapa Library Quantification Kit (Kapa Biosystems). The libraries were pooled and diluted to 2 nM in EB buffer (Qiagen). Before loading to an Illumina HiSeq 2000 sequencing analyzer (with TruSeq SR v3 flow cells), the libraries were denatured and diluted to 10 pM in pre-chilled hybridization buffer. After 75 cycles using a single-read recipe (TruSeq SBS Kit v3, Illumina), de-multiplexed sequencing reads (fastq files) that passed the default purify filter were obtained using the Illumina CASAVA pipeline (version 1.8).