Superscript 3 reverse transcriptase

Plant genomic DNA was isolated following previously described method40 (link).
Total RNA was isolated from 100 mg plant samples with Trizol reagent (Ambion, USA), and the genomic DNA is removed by using RNase-free DNase I (Invitrogen, USA). 2 μg total RNA was used to synthesize first strand cDNA with SuperScript III Reverse Transcriptase (Invitrogen, USA) according to manufacturer’s instructions. The first strand cDNA was used for semi quantitative RT-PCR and regular real-time PCR.
To determine the transcript level of mature miR395, the first-strand cDNA used for stem-loop real-time PCR was synthesized following the regular SuperScript III Reverse Transcriptase (Invitrogen, USA) mediated method, except that the oligo (dT)₂₀ was replaced with miR395 specific reverse transcription primer. Primers were all listed in Supplementary Table S1.

To determine the terminal sequence including poly(A) tail length for BCoV genomic RNA, a head-to-tail ligation method was employed as described previously [28 (link)]. The head-to-tail ligated RNA was used for reverse transcription (RT) with SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA) and PCR was performed with AccuPrime Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA) and oligonucleotides binding to 5′ and 3′ UTR of the genome followed by sequencing. The structures of the cis-acting RNA located in the 5′ UTR were predicted using the Mfold algorithm [29 (link)]. To determine whether the genome structures of BCoV were altered under the persistence, random hexamer oligonucleotides were used for RT with SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA), and the resulting cDNA was used for PCR with PfuUltra II high-fidelity DNA polymerase (Agilent, Santa Clara, CA, USA). The resultant PCR products were then subject to sequencing analysis. Because conventional Sanger sequencing was used, the sequences shown in the passaging experiments represented the genome structure of the main virus population but not the quasispecies. For the identification of the TRS employed for the synthesis of sgmRNAs, oligonucleotides binding to the leader sequence, and the sequence downstream of the TRS of each sgmRNA were used followed by sequencing.

To quantify s4/m3 mRNA, total RNA extracted from reovirus-infected cells were used for reverse transcription using SuperScript III Reverse Transcriptase (Invitrogen) without pre-denaturing steps. Random hexamer primer (Thermo Fisher SO142) was used for RT steps. After cDNA was generated, s4/m3 mRNA or housekeeping gene GAPDH was assessed by Applied Biosystems Fast SYBR Green Master Mix (Thermo Fisher 43–856012). Primers sequences are as follows: s4 forward: 5′-CGCTTTTGAAGGTCGTGTATCA; s4 reverse: 5′-CTGGCTGTGCTGAGATTGTTTT; m3 forward: 5′-CGTGGTCATGGCTTCATTC; m3 reverse: 5′-CACATGCTGATAAGGTATAGACAT; GAPDH forward: 5′-TGATGACATCAAGAAGGTGGTGAAG; GAPDH reverse: 5′-TCCTTGGAGGCCATGTAGGCCAT).
To examine replication of viral genome, we used a modified qPCR assay to quantify s4 negative-sense ssRNA which was described previously [105 (link)]. Briefly, total RNA extracted from reovirus-infected cells was denatured at 95°C for 3 min and subjected to reverse transcription using SuperScript III Reverse Transcriptase (Invitrogen). A primer (s4 forward: 5′-CGCTTTTGAAGGTCGTGTATCA) that only binds to the negative-sense strand of s4 was used to reverse transcribe s4 RNA to s4 negative-sense cDNA. Subsequent PCR was performed with both forward and reverse primers for s4 as described above. All results were normalized to T1L WT.

Following RNA extraction, cDNA synthesis was performed using 250 ng of RNA and SuperScript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). PCR was carried out using Q5 DNA polymerase according to the manufacturer’s instructions (New England Biolabs, Ipswich, MA, USA). Primers were either designed for this study or published before (Table S2) [45 (link),46 (link)]. PCR-produced fragments were cleaned and cloned in pGEM-T vector (Promega, Madison, WI, USA) using the manufacturer’s instructions, followed by sequencing. The resulting sequences were assembled and aligned using the CLC Free Workbench (https://digitalinsights.qiagen.com/products-overview/discovery-insights-portfolio/analysis-and-visualization/qiagen-clc-main-workbench/ accessed on 8 December 2021) and were then manually analyzed.
For the evaluation of the PSTVd titer in both the total RNA extract and the polysome fraction, cDNA was prepared by reverse transcribing 500 ng RNA (SuperScript III reverse transcriptase—Invitrogen, Carlsbad, CA, USA) in the presence of random primers. Three housekeeping genes, specifically the 5.8S, 18S, and 25S rRNAs, were used for normalization, and three biological and three technical replicates were used. The qBASE framework was used for the analysis [47 (link)].
The detection of PSTVd by northern blotting was carried out as described previously [34 (link),36 (link)].

The mRNA levels of DNMT1, BRCA1, and 36P4 in breast tissues were determined by quantitative reverse transcription polymerase chain reaction (qRT-PCR). The total RNA was isolated and purified from breast tissues using kits (Superscript III Reverse Transcriptase, Invitrogen, Waltham, MA, USA). The RNA was used to create cDNA libraries (Superscript III Reverse Transcriptase, Invitrogen) by using specific primers. RT-PCR was then carried out by employing SYBR Green qPCR (iQ SUPERMIX, BioRad, Hercules, CA, USA) on an ABI7500 system. The used qPCR conditions were as follows: 95 °C for 30 s, 60 °C for 40 s, and finally 72 °C for 40 s. The results were finally normalized to those obtained with 36P4 mRNA. The CT values of the samples were determined, and relative expression was calculated using the 2^−ΔΔCT method. The sequences of the primers used for the PCR amplification were as follows: DNMT1 (sense: 5′-GGCCTTTTCACCTCCATCAA-3′; antisense: 5′-GCACAAACTGACCTGCTTCA-3′); BRCA1 (sense: 5′-CCGCCTTGCTTTAACTGATGT-3′; antisense: 5′-CACTTTCCTCCTGCAATGCC-3′); 36P4 (sense: 5′-AGTACCTGCTCAGAACACCG-3′ antisense: 5′-GCCATTGTCAAACACCTGCT-3′). Amplicons were size controlled on agarose gel and purity was assessed by analysis of the melting curves at the end of the RT-PCR reaction.

To remove the free nucleic acid and eliminate the contaminating host genomic DNA, 14 U Turbo DNase (Ambion, Austin, TX, USA), 25 U Benzonase Nuclease (Novagen, San Diego, CA, USA), 20 U RNase I (Fermentas, Ontario, Canada), and 10 × DNase buffer (Ambion) were added to 127 μL of the supernatants to a final volume of 150 μL, followed by digestion at 37°C for 1 h. The viral nucleic acid in the obtained products was extracted using a virus nucleic acid isolation kit (Bioer Technology, Hangzhou, China) according to the manufacturer's instructions. The total viral nucleic acids were reverse-transcribed using anchored random primers and Superscript III reverse transcriptase (Invitrogen, Carlsbad, CA, USA). The anchored random primers (Table 2) were added separately to the viral nucleic acid and incubated at 75°C for 5 min and then placed on ice for 5 min for denaturation. To obtain the reverse-transcribed product, 40 U of RNase OUT (Invitrogen, Carlsbad, USA), 200 U of Superscript III reverse transcriptase (Invitrogen), 1 μL of 0.1 M dithiothreitol (DTT) (Invitrogen), 1 μL of 10 mM dNTPs (TaKaRa, Dalian China), 4 μL of 5 × first-strand buffer (Invitrogen), and RNase-free H₂O (TaKaRa) were added to a final volume of 20 μL and incubated at 25°C for 10 min, followed by 50°C for 60 min and then 75°C for 10 min.