RNA-Seq of HUVECs
Library preparation was performed using Optimal Dual-mode mRNA Library Prep Kit (BGI-Shenzhen, China). A certain amount of RNA were denatured at suitable temperature to open the secondary structure, and mRNA was enriched by oligo (dT) attached magnetic beads. After reacting at a suitable temperature for a fixed time period, RNAs were fragmented with fragmentation reagents. Then first-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. The synthesized double strand cDNA was subject to end repairment reaction. After cDNA end repairment, a single A nucleotide was added to the 3 ends of the blunt fragments through A tailing reaction. Then the reaction system for adaptor ligation configured to ligate adaptors with the cDNAs, and finally, the library products were amplified through PCR reaction and subjected to quality control. Next, the single-stranded library products were produced via denaturation. The reaction system for circularization was set up to get the single-stranded cyclized DNA products. Any uncyclized single stranded linear DNA molecules would be digested. The final single strand circularized library was amplified with phi29 and rolling circle amplification (RCA) to make DNA nanoball (DNB) which carried more than 300 copies of the initial single stranded circularized library molecule. The DNBs were loaded into the patterned nanoarray and SE50 bases reads were generated on G400 platform (BGI-Shenzhen).