GSM8993819: PDGF-BB, rep2; Homo sapiens; RNA-Seq
RNA from HASMCs lysed with TRIzol (Invitrogen, 15596026) was extracted using the RNeasy Micro Kit (QIAGEN, Germany) following the manufacturer's instructions. Briefly, TRIzol-lysed samples were thawed and mixed with 1/5 volume of chloroform/iso-amyl alcohol (24:1), then centrifuged at 12,000×g for 10 min. The supernatant was transferred to a new tube and mixed with an equal volume of 75% ethanol. The mixture was applied to an RNeasy MinElute spin column, followed by sequential washing with RW1 and RPE buffers. DNA digestion was performed using DNase I and Buffer RDD at room temperature for 15 min. Finally, RNA was eluted with 20 µL RNase-free water and quantified/qualified using a Fragment Analyzer, Agilent 2100 Bioanalyzer, or Qseq-400 with fluorescent dye-based methods. Library preparation is performed using Optimal Dual-mode mRNA Library Prep Kit (BGI-Shenzhen, China). A certain amount of RNA treated with Hieff NGS MaxUp Human rRNA Depletion Kit (Yeasen-Shanghai, China) to remove rRNA. After reacting at a suitable temperature for a fixed time period, RNAs are fragmented with fragmentation reagents. Then First-strand cDNA is generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis in a new reaction system containing dUTP. The synthesized double strand cDNA is subject to end repairment reaction. After cDNA end repairment, a single 'A' nucleotide is added to the 3' ends of the blunt fragments through A tailing reaction. Then the reaction system for adaptor ligation is configured to ligate adaptors with the cDNAs, and finally, the library products are amplified through PCR reaction and subjected to quality control. Next, the single-stranded library products are produced via denaturation. The reaction system for circularization is set up to get the single-stranded circularized DNA products. Any single stranded linear DNA molecules will be digested. The final single strand circularized library is amplified with phi29 and rolling circle amplification (RCA) to make DNA nanoball (DNB) which carries more than 300 copies of the initial single stranded circularized library molecule. The DNBs are loaded into the patterned nanoarray and sequencing reads of PE150 bases length are generated on T7 platform (BGI-Shenzhen, China).