GSM7594315: primary rat microvessels, stiff ECM, with A83-01; Rattus norvegicus; RNA-Seq
Total RNA was extracted from the microvascular cells using Trizol (Invitrogen, Cat#15596026) according to the manufacture instruction. The cells of a 60mm dish were homogenized in 1ml Trizol, centrifuged at 12,000×g for 5min at 4°C, and then the supernatant was transferred into a tube with 0.3ml chloroform/isoamyl alcohol (24:1). The mixture was centrifuged at 12,000×g for 10min at 4°C, the upper aqueous RNA layer was transferred into a new tube with equal volume of isopropyl alcohol, and then cen trifuged at 12,000×g for 20min at 4°C. The RNA pellet was washed with 1ml 75% ethanol, and then centrifuged at 12,000×g for 3min at 4°C. The pellet was dried in air for 5–10min. Finally, 50µl DEPC water was added to dis solve the RNA, which was qualified and quantified using a Nano Drop and Agilent 2100 bioanalyzer (ThermoFisher). The mRNA was purified by Oligo(dT)-attached magnetic beads and fragmented into small pieces. First-strand cDNA was generated by random hexamer-primed reverse tran scription and a second-strand cDNA synthesis. A-Tailing Mix and RNA Index Adapters were added. The cDNA fragments were amplified by PCR, and then purified by Ampure XP Beads and dissolved in EB solution. The product was validated on an Agilent Tech 2100 bioanalyzer. The double stranded PCR products were denatured and circularized by the splint oligo sequence to make the final library, which was amplified with phi29 to make DNA nanoball with more than 300 copies of one molecular, which were loaded into the patterned nanoarray and pair end 150 bases reads were generated on DNBSEQ-T7 platform.