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.本研究用于制备体外转录RNA（in vitro transcribed RNA, IVT RNA）的实验流程如下：先以加poly(A)尾的RNA为模板进行逆转录，合成双链cDNA，再将该双链cDNA用于后续体外转录反应。逆转录所用引物参照已发表的实验方案（文献17、50）。 本研究所用的以下寡核苷酸序列均购自中华人民共和国深圳BGI Genomics：模板转换寡核苷酸（template switching oligo, TSO）T7引物：5′-ACTCTAATACGACTCACTATAGGGAGAGGGCrGrGrG-3′，其中小写r代表核糖核苷酸碱基；T7延伸引物：5′-GCTCTAATACGACTCACTATAGG-3′。 逆转录反应条件参照已发表文献（文献51）。具体反应体系为：将总质量300 ng的RNA溶于6 μl无核酸酶水中，先与1 μl浓度为10 μM的oligo(dT)23VN引物（New England Biolabs）及1 μl浓度为10 mM的dNTP混合，于72℃孵育3分钟、4℃孵育10分钟、25℃孵育1分钟，热盖温度设置为≥85℃，随后维持在4℃。 随后配制逆转录（reverse transcription, RT）混合液：包含3 μl无核酸酶水、4.4 μl 5×逆转录缓冲液、1 μl RNaseOut（Invitrogen）、1 μl浓度为75 μM的TSO T7引物，以及1 μl Maxima H Minus逆转录酶（Thermo Scientific）。将上述逆转录混合液加入退火后的RNA体系中，随后按照SSIV逆转录实验方案（文献51）进行孵育反应。 随后按照生产商说明书，使用耐热RNase H（New England Biolabs）水解RNA模板。采用VAHTS RNA清洁磁珠对模板转换cDNA产物进行纯化。 冰上配制第二链cDNA合成反应混合液：包含20 μl模板转换cDNA、25 μl Q5 Hot Start高保真预混液（New England Biolabs）、3.75 μl浓度为50 μM的T7延伸引物，以及1.25 μl无核酸酶水。反应体系先经95℃变性1分钟、57℃退火30秒，随后于65℃孵育10分钟。采用1倍体积的AMPure XP磁珠（Beckman Coulter）对所得双链DNA（double-strand DNA, dsDNA）进行纯化。 体外转录步骤采用MEGAscript转录试剂盒（Ambion，美国马萨诸塞州），于37℃反应4小时。本研究中制备的IVT RNA特指无修饰RNA，因此反应混合液组成为：各2 μl的NTP、2 μl反应缓冲液、溶于8 μl无核酸酶水中的120 ng双链cDNA模板，以及2 μl酶混合液。