Goscript 5 reaction buffer

Total RNA from the cell samples was reverse-transcribed using a First-Stand complementary DNA (cDNA) synthesis kit according to the manufacturer instructions (Promega, Madison, WI, USA). For reverse transcription (RT), 3 µg of the total RNA was mixed with 1 µL of Oligo (dT)₁₅ (0.5 µg/reaction) and nuclease-free water and heated in a 70 °C heat block for five minutes. After pre-incubation, the reverse transcription reaction mix containing: 4 µL GoScript 5× reaction buffer (Promega, Madison, WI, USA), 3µL MgCl₂ (final concentration 1.5–5.0 mM), 1 µL dNTPs (10 mM), 1 µL Recombinant RNases Ribonuclease Inhibitor (20 U/µL), and 1 µL GoScript Reverse Transcriptase was prepared.

Total RNA from the cell samples was reverse-transcribed using the GoScript™ Reverse Transcriptase according to the manufacturer’s instructions (Promega Corporation). For reverse transcription (RT), 3 µg of total RNA was mixed with 1 µl of Oligo d(T) primer (0.5 µg/μl) and water pretreated with diethylpyrocarbonate (H₂O-DEPC) and incubated for 5 min at 70 °C. After preincubation, other components were added to this mixture: 4 µl GoScript™ 5× Reaction buffer (Promega Corporation), 3 µl MgCl₂, 1 µl RNase inhibitor (20 U/µl), 1 µl deoxyribonucleotide triphosphates (dNTPs, 10 mM), and 1 µl GoScript™ Reverse Transcriptase (160 U/µl) in a total volume of 20 µl. The mixture was first incubated for 5 min at 25 °C, then for 60 min at 42 °C, and for the final 15 min at 70 °C, and stored at −20 °C.

0.5 μg of cytoplasmic RNA was spiked with 1 fmol of CleanCap^® mCherry mRNA (Trilink L7023) and 0.5 μl of oligo(dT)₁₅ primer (500 μg/ml) in a total volume of 10 μl. The mixture was incubated at 65°C for 5 min and immediately placed on ice. The mixture was brought to 20 μl with 4 μl of 25 mM MgCl₂, 1 μl of 10 mM dNTPs, 4 μl of GoScript^® 5× Reaction Buffer and 1 μl of GoScript^® Reverse Transcriptase (Promega A2791). Reactions were placed in the thermocycler at 25°C for 5 min, 42°C for 1 h and 70°C for 15 min. The resulting cDNA was quantified by real time PCR in technical triplicate reactions containing 0.5 μM reverse and forward primer (Supplementary Table S4) and 1× SensiFAST SYBR No-ROX (Bioline, BIO-98005) with a Bio-Rad CFX Connect real-time PCR detection system. PCR was performed with the protocol of 95°C for 3 min, 40 cycles of (95°C for 10 s, 55°C for 30 s).