title: GSM6755794: biol rep 3: SLE_S_high_3; Homo sapiens; RNA-Seq
library_construction_protocol: Total RNA was extracted from the PBMC using Trizol (Invitrogen, Carlsbad, CA, USA) according to manual instruction. PBMC were ground into powder by liquid nitrogen in a 2 mL tube, followed by being homogenized for 2 minutes and rested horizontally for 5 minutes. The mix was centrifuged for 5 minutes at 12,000×g at 4°C, then the supernatant was transferred into a new EP tube with 0.3 mL chloroform/isoamyl alcohol (24:1). The mix was shacked vigorously for 15s, and then centrifuged at 12,000×g for 10 minutes at 4°C. After centrifugation, the upper aqueous phase where RNA remained was transferred into a new tube with equal volume of supernatant of isopropyl alcohol, then centrifuged at 13,600 rpm for 20 minutes at 4°C. After   deserting the supernatant, the RNA pellet was washed twice with 1 mL 75% ethanol, then the mix was centrifuged at 13,600 rpm for 3 minutes at 4°C to collect residual ethanol, followed by the pellet air dry for 5-10 minutes in the biosafety cabinet. Finally, 25µL~100µL of DEPC-treated water was added to dissolve the RNA. Subsequently, total RNA was qualified and quantified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA) Limited RNA（more than 200pg, high-quality）was amplified with oligo-dT and dNTPs, incubated at 72℃ and immediately put back on ice, then reverse transcribed to cDNA based on polyA tail. The template was switched to the 5' end of the RNA and the fulllength cDNA was generated by PCR. Agilent 2100 bioanalyzer instrument (Thermo Fisher Scientific, MA, USA) was used to determine the average molecule length of PCR product. Purified cDNA from previous steps was fragmented into small pieces with fragment buffer by PCR, and the product was purified and selected by the Agencourt AMPure XP-Medium kit (Thermo Fisher Scientific, USA). cDNA was quantified by Agilent Technologies 2100 bioanalyzer. The double stranded PCR product undergo QC step was heat denatured and circularized by the splint oligo sequence. The single strand circle DNA (ssCir DNA) was formatted as the final library.  The final library was quantitated in two ways in order to ensure the high quality of the sequencing data: Determined the average molecule length used the Agilent 2100 bioanalyzer instrument, quantified library used real-time quantitative PCR (qPCR).The final library was amplified with phi29(Thermo Fisher Scientific, MA, USA) to make DNA nanoball (DNB) which had more than 300 copies of one molecular , DNBs were loaded into the patterned nanoarray and pair end 100 bases reads were generated on DNBSEQ G400 platform(BGI-Shenzhen, China) .