Instrument: MGISEQ-2000RS
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: PAIRED
Construction protocol: Total RNA was extracted from cells using Trizol (Invitrogen, Carlsbad, CA, USA) according to the manual instructions. Cells were washed with PBS and the 1.5ml Trizol reagent was added into the 2ml tube containing the cell pellets. The mix was centrifuge at 12000×g for 5min at 4°C. The supernatant was transferred to a new 2.0ml tube which was added 0.3 ml of Chloroform/isoamyl alcohol (24:1) per 1.5ml of Trizol reagent. After the mix was centrifuged at 12000×g for 10min at 4°C, the aqueous phase was transferred to a new 1.5mLtube which was add equal volume of supernatant of isopropyl alcohol. The mix was centrifuged at12000×g for 20min at 4°C and then removed the supernatant. After washed with 1 ml 75% ethanol, the RNA pellet was air-dried in the biosafety cabinet and then dissolved by add 25µL~100µL of DEPC-treated water. Subsequently, total RNA was qualified and quantified using a Nano Drop and Agilent 2100 bioanalyzer (Thermo Fisher Scientific, MA, USA). According to the manufacturer's instructions, the first step involves the removal of ribosomal RNA (rRNA) using target-specific oligos and RNase H reagents to deplete both cytoplasmic (5S rRNA,5.8S rRNA, 18S rRNA and 28S rRNA) and mitochodrial ribosomal RNA (12S rRNA and 16SrRNA) from total RNA preparations. Following SPRI beads purification, the RNA is fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments are copied into first strand cDNA using reverse transcriptase and random primers, followed by second strand cDNA synthesis using DNA Polymerase I and RNase H. This process removes the RNA template and synthesizes a replacement strand, incorporating dUTP in place of dTTP to generate ds cDNA. These cDNA fragments then have the addition of a single 'A' base and subsequent ligation of the adapter. After UDG treatment, the incorporation of dUTP quenches the second strand during amplification. The products are enriched with PCR to create the final cDNA library. The libraries were assessed quality and quantity in two methods: check the distribution of the fragments size using the Agilent 2100 bioanalyzer, and quantify the library using real-time quantitative PCR (QPCR) (TaqMan Probe). The qualified libraries were sequenced pair end on the BGISEQ-500/ MGISEQ-2000 System (BGI-Shenzhen, China).