Iscript reverse transcription supermix

Cells were lysed in RA1 buffer supplemented with 1% (v/v) β-mercaptoethanol (Macherey-Nagel) and stored at −80°C until processing. Total RNA was extracted by using the NucleoSpin RNA Extraction Kit (Macherey-Nagel) and the residual genomic DNA was removed by DNase digestion with RNase-free DNase (Macherey-Nagel). The total RNA quantifications were carried out using a NanoDrop spectrophotometer (NanoDrop Technologies). For cDNA synthesis, total RNA (100 ng) was reverse transcribed using the 5X iScript reverse transcription supermix (Biorad) according to the manufacturer's instructions. cDNA samples were stored at −20°C until use. Reverse transcription-quantitative PCR assays were performed in a LightCycler 480 instrument (Roche). Four microliter of 10-fold diluted cDNA were added to a mixture of (2X) iTaq Universal SYBR Green Supermix (Biorad) and 0.25 μM of each primer in a total volume of 10 μl. Thermal protocol was 95°C for 5 min followed by 40 cycles of 95°C for 10 s, 60°C for 30 s, and acquisition of a melting curve at the end of the run. The specificity of primer pairs was checked via melting curve analysis. Primers used in this study (Supplementary Table 1) were designed and tested as previously described (14 (link)). Fold changes were calculated by the ΔΔCt method using ACTB, PPIA, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) as reference genes (15 (link)).

RNA was extracted from infected cell monolayers with TRIzol Reagent (Thermo Fisher Scientific-Ambion, Carlsbad, CA, USA) following the manufacturer’s instructions and stored at −80 °C. The quality, purity, and concentration of the RNA were measured using a NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Wilmington, DE, USA). cDNA was synthesized using 5X iScript Reverse Transcription Supermix (Bio-Rad, Hercules, CA, USA) to yield 20 µL of cDNA for every 250 ng of RNA template. Reverse transcription was performed in the T100 Thermocycler (Bio-Rad) under the following conditions: priming for 5 min at 25 °C; reverse transcription for 30 min at 42 °C; and inactivation for 5 min at 85 °C. cDNA was diluted to 1:10 with nuclease-free water and stored at −20 °C for later use.

Cells (2000–5000) were sorted directly into Trizol LS (Thermo Fisher Scientific). Total RNA was extracted and reverse-transcribed using iScript reverse transcription supermix (Bio-Rad). Real-time PCR was performed using the iTaq Universal SYBR Green supermix (Bio-Rad) using a CFX384 real-time PCR machine (Bio-Rad). Transcript levels were normalized to Actin (Actb) using the ΔCt method. The primers used for qPCR analysis are listed in Supplemental Table S3.