design: Two tubes of blood were collected twice 12 hours apart (7:00-9:00 and 19:00-21:00) by venipuncture in either arm. A 3mL TempusTM Blood RNA tube (Applied Biosystems, Foster City, CA) was used to collect stabilized whole blood RNA. RNA was extracted using Tempus Spin RNA Isolation kits, according to manufacturers protocol. RNA integrity (7.5) and concentration were assessed prior to library preparation, with globin clearance. Library preparation was performed using Optimal Dual-mode mRNA Library Prep Kit (BGI-Shenzhen, China). RNA was taken to prepare the reaction system. The globin mRNA was removed by using GLOBINclear Whole Blood Globin Reduction Kit-Human (Ambion, USA). Then, the globin mRNA removed RNA samples 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, the enriched mRNA was fragmented with fragmentation reaction. Then the first-strand cDNA was generated using random hexamer-primed reverse transcription, followed by a second-strand cDNA synthesis. Related, 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 adaptors were connected to the double stranded cDNA with adaptor ligation reaction. 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 circularized DNA products. Any single stranded linear DNA molecules will be digested. The final single strand circularized library was 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 were loaded into the patterned nanoarray and sequencing reads of PE 150 bases length were generated on G400 platform (BGI-Shenzhen, China).