IVT_neg_004

For the generation of in vitro transcribed (IVT) RNA, the poly(A)-tailed RNA underwent reverse transcription to synthesize double-stranded cDNA, which was then used for in vitro transcription. Primers used in reverse transcription referred to previously published protocols (17, 50). The following oligonucleotides were purchased from the BGI Genomics, SZ, PRC: template switching oligo (TSO) T7 primer, 5′-ACTCTAATACGACTCACTATAGGGAGAGGGCrGrGrG-3′, where r indicates ribonucleotide bases; T7 extension primer, 5′-GCTCTAATACGACTCACTATAGG-3′. For the reverse transcription conditions, we referred to a previously published literature (51). In total, 300 ng RNA in 6 μl nuclease-free water was first annealed with 1 μl oligo(dT)23VN primer (10 μM, New England Biolabs) and 1 μl dNTP (10 mM, New England Biolabs) for 3 min at 72°C, 10 min at 4°C, 1 min at 25°C with the lid temperature set at ≥85°C and then held at 4°C. The reverse transcription (RT) mix was assembled containing 3 μl nuclease-free water, 4.4 μl 5xRT Buffer, 1 μl RNaseOut (Invitrogen), 1 μl TSO T7 primer (75 μM), and 1 μl Maxima H Minus Reverse Transcriptase (Thermo Scientific). After adding the RT mix to the annealed RNA, the reaction mixture was incubated following the SSIV RT protocol (51). The RNA template was subsequently hydrolyzed by the Thermostable RNase H (New England Biolabs) according to the manufacturer's instructions. The template-switching cDNA product was purified using the VAHTS RNA Clean Beads. The second-strand cDNA synthesis reaction mixture was assembled on ice consisting of 20 μl template-switching cDNA, 25 μl Q5 Hot Start High Fidelity Master Mix (New England Biolabs), 3.75 μl T7 extension primer (50 μM), and 1.25 μl nuclease-free water. Following the initial denaturation at 95°C for 1 min and 57°C for 30 sec for annealing, the reaction mixture was incubated at 65°C for 10 min. The resulting double-strand DNA (dsDNA) was purified using 1 volume of AMPure XP beads (Beckman Coulter). The in vitro transcription step was performed using the MEGAscript Kit (Ambion, MA, USA) at 37°C for 4 h. IVT RNA prepared in this article refers specifically to modification-free RNA, so the reaction mixture was composed of 2 μl each of NTPs, 2 μl Reaction Buffer, 120 ng ds-cDNA template in 8 μl nuclease-free water, and 2 μl Enzyme Mix.