Reverse transcription kit

Total cellular RNA was extracted from HBVSMC according to the manufacturer's protocol (Qiagen RNeasy Mini Kit, Qiagen, Hilden, Germany). Total RNA was subsequently converted into cDNA by Qiagen Reverse Transcription Kit (Qiagen, Hilden, Germany). Real-time PCR was carried out using the 7500 Real-Time PCR system (Applied Biosystems) under standard conditions with 200 pM PCR primers. Each sample was analyzed in triplets using SYBR green (Applied Biosystems) as fluorescent detector and GAPDH as endogenous control. All primers were obtained from RT qPCR Primer assay kits (SAB Bioscience).

Total RNA was extracted from whole brain tissue (left and right hemisphere) according to the manufacturer's protocol (Qiagen RNeasy Mini Kit, Qiagen, Hilden, Germany). Total RNA was reverse transcribed into cDNA by Qiagen Reverse Transcription Kit (Qiagen, Hilden, Germany). Real-time PCR was carried out using 7500 Real-Time PCR system (Applied Biosystems) with 200 pM PCR primers. Each sample was analyzed using SYBR green (Applied Biosystems) as fluorescent detector and actin as endogenous control. All primers were obtained from RT qPCR Primer assay kits (SAB Bioscience). The data were analyzed and expressed as fold using comparative Ct method.

Total RNA was extracted in YPD, YPD + 7% ethanol and YP + 2% glycerol media by either using Qiagen RNeasy Mini kit for real time PCR experiments (catalogue no. 74104) or by using Trizol reagent for RNA sequencing (Invitrogen, catalogue no. 155-96-018). cDNA from total RNA was synthesized using Qiagen reverse transcription kit (catalogue no. 205311). The expression level of IFA38 was determined using quantitative real-time PCR as described previously [34 (link)]. The primers used for the real-time PCR are in electronic supplementary material, table S4.

Total RNA was isolated using a RNeasy plus mini kit (Qiagen, Germantown, MD, USA) 12 h after transfection. Then, a Qiagen reverse transcription kit was used for reverse transcription. A pair of specific primers, PAX9-F: 5′-AACCAGCTGGGAGGAGTGTT-3′ and PAX9-R: 5′-TGATGTCACGGTCGGATG-3′, were designed. They are located at the N-terminal of the paired box domain and can identify wild type and variant mRNA. The expression of PAX9 was normalized by eukaryotic 18S ribosomal RNA (rRNA). Cells were treated with 10 μg/mL actinomycin D (Gibco) 12 h after transfection. After 4 and 8 h of treatment, total RNA was harvested for reverse transcription. The stability of mRNA is expressed as the percentage of remaining PAX9 mRNA, as measured by 18SrRNA.

Total RNA from top 10 selected PYC2-CHO clones including CHO-S cells (parental cell) as a control was extracted using the Qiagen RNA extraction kit and the manufacturer's protocol. RNA concentration was determined by using nano-drop spectrophotometer, and its quality and integrity were checked by electrophoresis on 1.5% (w/v) agarose gel. The RNA was treated with DNaseI (Thermo Fisher Scientific, USA) prior to cDNA synthesis as per manufacturer’s recommended. For reverse transcription (RT), we used 500ng of total RNA transcribed into cDNA in a 20μl reaction mixture performed using Qiagen reverse transcription kit and the oligo (dT)15 primers provided in the same kit. To check the PYC mRNA expression, gene-specific forward and reverse oligonucleotides were used (synthesized from Sigma Aldrich, USA) in a 20μl reaction mixture and carried out PCR for 40 cycles [Initially 95°C for 7min and cycle repeated for 95°C-15 second and 60°C (annealing and extension) for 30 seconds (Pikoreal, Thermo fisher Scientific, USA). Quantification was carried out using formula relative quantification method 2^{- ΔΔCq}[51 (link)]. The PCR product was then electrophoresed on 1% (w/v) agarose gel to confirm the PYC2 expression of top 10 selected clones.