GSM8896477: 375; Homo sapiens; RNA-Seq
Total RNA was extracted using Trizol reagent (Invitrogen, Carlsbad, CA, USA). Samples were frozen in a 2mL tube using liquid nitrogen, homogenized in 1.5ml of TRIZOL reagent for 2 min, and then allowed to rest horizontally for 5 min. After centrifugation at 12000g for 5 min at 4°C, the supernatant was collected and mixed with 0.3 mL chloroform/isoamyl alcohol (24:1). The mixture was vigorously shaken for 15 s and then centrifuged at 12000g for 10 min at 4°C. After centrifugation, the upper aqueous phase was mixed with an equal volume of isopropyl alcohol and was centrifuged at 13600g for 20 min at 4°C. After deserting the supernatant, the RNA pellet was washed twice with 1 mL of 75% ethanol and air-dried for 5-10 min. RNA samples were dissolved in 25-100μL of DEPC-treated water. Total RNA was qualified and quantified using a NanoDrop (Thermo Fisher Scientific, MA, USA) and Agilent 2100 bioanalyzer (Agilent, CA, US). Oligo (dT)-attached magnetic beads were utilized to purify mRNA. Purified mRNA was fragmented into small pieces with fragment buffer (75mM KCl, 6mM MgCl2, 50mM Tris-HCl, pH 8.3). First-strand cDNA was generated using random hexamer-primed reverse transcription, followed by second-strand cDNA synthesis. Afterward, a-Tailing Mix and RNA Index Adapters were added for end repair. The cDNA fragments were amplified by PCR and the resulting PCR products were purified by Ampure XP Beads. The purified products were dissolved in EB (10mM Tris-HCl, pH8.5) solution and were validated on the Agilent 2100 bioanalyzer for quality control. The double-stranded PCR products from the previous step were heat-denatured and circularized by the splint oligo sequence to generate the final library as single-strand circular DNA. The final library was amplified with phi29 to produce DNA nanoballs (DNB), each containing more than 300 copies of single molecule. The DNBs were then loaded into the patterned nanoarray, and paired-end 150 base reads were generated by BGISEQ-500.