Total (input) or AGO-immunoprecipitated RNA was reverse transcribed82 (link). Briefly, 100–500 ng of input or IP RNA was reverse transcribed in a final volume of 10 µL containing 2 µL 5× ProtoScript II reaction buffer (NEB), 25 µM ATP, 25 µM dNTPs, 50 µM RT primer (IK-44), 1 unit of poly(A) polymerase (Invitrogen) and 20 units of protoscript II reverse transcriptase (NEB). Reactions were incubated at 42 °C for 1 h followed by enzyme inactivation at 95 °C for 5 min. Real-time quantitative reverse transcriptase PCR (RT qPCR) was performed using a LightCycler 480 II (Roche) with SensiFAST SYBER No-ROX (Bioline Meridian Biosystems) using the gene-specific primers listed in Supplementary Table 2 . PCR was carried out in technical triplicates using the following cycling conditions: 95 °C for 3 min, followed by 45 cycles of denaturation at 95 °C for 10 s, annealing at 60 °C for 10 s, and elongation at 72 °C for 20 s. A melting curve was generated at the end of the amplification in every run to confirm primer specificity. Threshold cycle (Ct) values were determined by calculating the second derivative maximum of three technical triplicates for each sample. Data were analysed using Prism-GraphPad Software v8.4.0.
Protoscript 2 reaction buffer
Manufactured by New England Biolabs
The ProtoScript II reaction buffer is a buffer solution designed for use in reverse transcription reactions. It provides the necessary ionic conditions and cofactors to facilitate the conversion of RNA to complementary DNA (cDNA) by reverse transcriptase enzymes.
Automatically generated - may contain errors
Lab products found in correlation
Poly a polymerase, by Thermo Fisher Scientific (1 mentions)
Rt primer, by Thermo Fisher Scientific (1 mentions)
Lightcycler 480 2, by Roche (1 mentions)
Protoscript 2 reverse transcriptase, by New England Biolabs (1 mentions)
Monarch pcr dna cleanup kit, by New England Biolabs (1 mentions)
Q5 high fidelity 2x master mix, by New England Biolabs (1 mentions)
Ribonuclease a, by Merck Group (1 mentions)
Protoscript 2, by New England Biolabs (1 mentions)
E coli dna ligase, by New England Biolabs (1 mentions)
E coli dna polymerase, by New England Biolabs (1 mentions)
Murine rnase inhibitor, by New England Biolabs (1 mentions)
E coli dna ligase, by Thermo Fisher Scientific (1 mentions)
Rnase h, by New England Biolabs (1 mentions)
Rnase h, by Thermo Fisher Scientific (1 mentions)
3 protocols using protoscript 2 reaction buffer
1
Reverse Transcription and RT-qPCR Protocol
2
Reverse Transcription Activity of Partitiviral RdRp
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To analyze the RT activity of RdRp recombinant proteins of partitiviruses, cDNA synthesis was performed using 100 ng RNA (transcript of PCV2 RdRp), 1 mM dNTPs, 1 μM reverse primer (PCV2-RdRp-T7-R, supplementary table 1 , Supplementary Material online), 1x ProtoScript II Reaction buffer (New England Biolabs), and 2 μM of recombinant proteins (as RT enzyme) in 10 μl total reaction volume. In the positive control reaction, 0.5 μl of ProtoScript II was added to the mixture as reverse transcriptase. The reactions without a template or RT enzyme are considered as two negative controls. The reactions with recombinant proteins and ProtoScript II were incubated for 3 h at 27°C and 42°C, respectively. After cDNA synthesis, 1 μl (10 mg/ml) of boiled ribonuclease A (Sigma, USA) was added to the reactions and incubated for 15 min at room temperature. The samples then were heated to 85°C for 2 min, and single-stranded cDNA was purified using a Monarch PCR & DNA clean-up kit (New England Biolabs) according to the manufacturer’s protocol. The cleaned cDNA was amplified by PCR using Q5 High-Fidelity 2X Master Mix (New England Biolabs) according to the manufacturer’s instructions. The activity and fidelity of RdRp protein (ACDMV RdRp) as an RT enzyme was confirmed by direct DNA sequencing of full-length PCR product of enzyme reaction.
3
Modified CEL-Seq2 with Reduced Volumes
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The CEL-Seq2 protocol with reduced volumes was used as previously described (Herman et al., 2018) and modified using the following reagents.
Instead of 1.2 μl vapor lock as hydrophobic encapsulation barrier mineral oil (Sigma, M8410-100ML) was used. For cDNA first-strand synthesis, Protoscript II and Protoscript II Reaction Buffer (NEB, M0368L) as well as murine RNase-Inhibitor (NEB, M0314S) was used instead of SuperScript II reverse transcriptase, first-strand synthesis buffer and RnaseOUT. Escherichia coli DNA polymerase I, E. coli DNA ligase, RNase H (Invitrogen; 18,021,071) and 5 × second-strand buffer were replaced with E. coli DNA polymerase (NEB, M0209L), E. coli DNA ligase (NEB, M0205L), RNaseH (NEB, M0297S), and 10 × Second-Strand Buffer (NEB, B6117S) respectively.
The water volume was adjusted to adequately dilute the 10x second-strand buffer. After second-strand synthesis, 96 wells were pooled, which results in 96 single cells per library.
The library preparation was performed as previously described [43 ], but by using Protoscript II, Protoscript II Reaction Buffer, and murine RNase-Inhibitor as mentioned above instead of SuperScript II reverse transcriptase, first-strand synthesis buffer, and RnaseOUT.
Instead of 1.2 μl vapor lock as hydrophobic encapsulation barrier mineral oil (Sigma, M8410-100ML) was used. For cDNA first-strand synthesis, Protoscript II and Protoscript II Reaction Buffer (NEB, M0368L) as well as murine RNase-Inhibitor (NEB, M0314S) was used instead of SuperScript II reverse transcriptase, first-strand synthesis buffer and RnaseOUT. Escherichia coli DNA polymerase I, E. coli DNA ligase, RNase H (Invitrogen; 18,021,071) and 5 × second-strand buffer were replaced with E. coli DNA polymerase (NEB, M0209L), E. coli DNA ligase (NEB, M0205L), RNaseH (NEB, M0297S), and 10 × Second-Strand Buffer (NEB, B6117S) respectively.
The water volume was adjusted to adequately dilute the 10x second-strand buffer. After second-strand synthesis, 96 wells were pooled, which results in 96 single cells per library.
The library preparation was performed as previously described [43 ], but by using Protoscript II, Protoscript II Reaction Buffer, and murine RNase-Inhibitor as mentioned above instead of SuperScript II reverse transcriptase, first-strand synthesis buffer, and RnaseOUT.
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