Oligo dt 15 primer

Total RNA was extracted from PAMs using TRIzol reagent (Magen, China) according to the manufacturer's instructions. Briefly, Reverse Transcription System (A3500, Promega, USA) was used for reverse transcription in 20 μL reaction volume. The reverse-transcription primers were Oligo (dT) 15 primer (C110A, Promega, USA) and Random primer (C118A, Promega, USA). SYBR Green (TaKaRa, Osaka, Japan) real-time PCR was performed using a Light-Cycler 480 PCR system (Roche, Basel, Switzerland). Relative quantities of mRNA accumulation were evaluated using the 2^−ΔCt method. The primers used for qRT-PCR are listed in Table 1.

The antennae from three-day-old virgin adults were dissected and immediately collected into a 1.5 mL Eppendorf tube, containing liquid nitrogen, and stored at −80°C until use. Total RNA was extracted by QIAzol Lysis Reagent following the manufacturer’s protocol (including DNase I treatment). RNA quality was checked with a spectrophotometer (NanoDrop 2000, Wilmington, DE). The single-stranded cDNA templates were synthesized using 2 μg total RNAs from various samples with 0.5 μg oligo (dT) 15 primer (Promega, Madison, WI), heated to 70°C for 5 min to melt the secondary structure within the template, then using M-MLV reverse transcriptase (Promega) at 42°C for 1 hr, and stored at −20°C.

Retinal total RNA was extracted using the TriFast^TM reagent (PeqLab, Erlangen, Germany) according to the manufacturer’s instructions. Residual genomic DNA contamination was eliminated by treatment with DNaseI (Amplification Grade; Invitrogen, Darmstadt, Germany). The first-strand cDNA synthesis was carried out using 1 μg of total RNA, 1× first-strand buffer (Invitrogen), Oligo(dT)15 primer (20 ng/μl; Promega, Mannheim, Germany), dNTPs (0.4 mM each; Carl Roth, Karlsruhe, Germany), RiboLock RNase Inhibitor (1.6 U/μl; Thermo Fisher Scientific, Schwerte, Germany) and SuperScript III reverse transcriptase (8 U/μl) according to the manufacturer’s manual. Forty nanogram of the transcribed cDNA were subsequently used as PCR template in reaction buffer (Qiagen, Hilden, Germany) containing MgCl₂ (1.5 mM), 0.2 mM dNTPs (Carl Roth), 0.4 μM primer and HotStar Taq polymerase (0.5 U/μl; Qiagen). The quality of the cDNA was tested using intron-spanning primers for β-actin (usp: 5′-tgttaccaactgggacgaca-3′; dsp: 5′-aaggaaggctggaaaagagc-3′; product size: 573 bp for cDNA and 1027 bp for gDNA). To amplify partial Cx30.2 cDNA, a specific primer set (usp: 5′-atgcaccaggccagcaaggag-3′; dsp: 5′-ccgcgctgcgatggcaaagag-3′; product size: 422 bp) and 1× Q-solution (Qiagen) was used.

Total RNA was isolated from CRISPR-targeted cells using a Direct-zol RNA Miniprep kit (Zymo Research), and 1 μg of total RNA was reverse transcribed using a reverse transcription system product with oligo(dT)15 primer (Promega, Fitchburg, WI). RReverse transcription-quantitative PCR (RT-qPCR) was performed using SYBR Green PCR Master Mix (Thermo Fisher Scientific, Waltham, MA).
DEPDC5 primers: 5′-GCTGTGAATGGTTTCCTTGCT-3′ and 5′-CTGTCGAATTGAGGCTCGGT-3′. TSC1 primers: 5′-ACCAGCCCTTATGCTGACAC-3′ and 5′-TTATCAGCCGTGTCGATGGG-3′. GAPDH primers: 5′-ACGGATTTGGTCGTATTGGG-3′ and 5′-ATCTCGCTCCTGGAAGATGG-3′. The PCR procedures were as follows: 95 °C for 5 min, followed by 40 cycles at 95 °C for 15 s and 60 °C for 30 s. Quantified mRNA was normalized to GAPDH as a control. The relative expression of mRNA was determined by the 2^ΔΔCT method according to the manufacturer’s instructions (Eppendorf, Hamburg, Germany).

Total RNA from GFP‐positive and mCherry‐positive cells (i.e., transfected cells and infected cells) and GFP‐negative and mCherry‐negative cells (i.e., nontransfected cells and noninfected cells) was purified using the Nucleopsin XS kit (Macherey‐Nagel). RNA were extracted as described in the manufacturer's manual and suspended in nuclease‐free water. The Superscript™ II First Strand Synthesis System (Invitrogen), with oligo (dT)15 primer (Promega), was used to synthetise cDNA. Quantitative RT‐PCR was carried out using the IQ™ SYBR Green Supermix (Qiagen). The qPCR was performed using the following protocol: 95°C for 3 min and 40 cycles at 95°C for 10 s and 60°C for 30 s followed by 65°C for 30 s. The melting curve was generated at 65°C for 5 s followed by gradual heating (0.5°C/s) to 95°C. BAX, BCL₂, and P21 gene expression values were normalised to the human housekeeping β‐actin (actin) and glyceraldehyde 3‐phosphate dehydrogenase (GAPDH) transcripts and the avian housekeeping β‐actin, G10 and GAPDH transcripts (see primers in Table S3). The relative gene expression levels were determined using the 2^(−Δct) method, and each experiment was performed in triplicate.