Taq pcr mastermix 2

About 0.1 g of fresh mycelium was collected and ground in liquid nitrogen. Total RNAs were extracted using RNA Easy Fast Plant Tissue Kit (Tiangen, Beijing, China). For the RT reaction, 10 μL of RT master mixture containing 6 μL ddH₂O, 3 μL 5× RT-PCR buffer, 1 μL dNTP mix, 0.5 μL reverse primer (OMSV-R), 0.25 μL M-MLV reverse transcriptase, and 0.25 μL RNase Inhibitor was added to 5 μL RNA. Reverse transcription was carried out at 37 °C for 90 min. The final volume of PCR was 15 µL, containing 0.2 µM each primer (OMSV-F/OMSV-R), 1× Taq PCR MasterMix II (Tiangen, Beijing, China), and 2 µL cDNA template. PCR cycling was performed as follows: 95 °C for 3 min, then 34 cycles of amplification with 95 °C for 30 s, 53 °C for 50 s, and 72 °C for 30 s, and a final extension at 72 °C for 10 min. For multiplex PCR, the program was set as follows: 95 °C for 5 min, followed by 30 cycles of 3 steps including 95 °C for 30 s, 62 °C for 30 s, 72 °C for 60 s, and a final extension at 72 °C for 10 min [48 (link)]. Amplification products were electrophoresed at 125 V for 40 min in 1% agarose gel.

All PCRs were performed on the PCR system (Bio-Rad, Hercules, CA, USA) in a 10 µL reaction volume containing 2 µL (20 ng/μL) of genomic DNA, 3 µL of 2 × Taq PCR Master Mix II (Tiangen, Beijing, China), 2 µL of forward and reverse primer mixture, and 3 µL of double distilled water (ddH₂O). The thermocycling conditions were as follows: 94 °C for 3 min; 6 cycles of 94 °C for 45 s, 55–65 °C for 1 min, and 72 °C for 1 min; 9 cycles of 94 °C for 45 s, 50–58 °C for 1 min, and 72 °C for 1 min; 19 cycles of 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min; and final extension at 72 °C for 5 min. Amplification products were analyzed by electrophoresis on 8.0% (w/v) denaturing polyacrylamide gel in TBE buffer for 1 h using the DYY-6C electrophoresis apparatus (Beijing Liuyi Instrument Factory, Beijing, China) under a constant voltage of 220 V. DNA fragments were then visualized by silver staining (Silver sequence staining reagents, Promega, Madison, WI, USA) and sized with 50 bp DNA ladder (Tiangen, Beijing, China) [51 (link)]. SSR primer sequences are listed in Table S1.

PCR was performed on the PCR system (Bio-Rad, Hercules, CA, USA) in a 10 µl reaction volume containing 2 µl (20 ng/µl) of genomic DNA, 3 µl of 2× Taq PCR Master Mix II (Tiangen, China), 2 µl of forward and reverse primer mixture, and 3 µl of ddH₂O. The thermocycling conditions were as follows: 94 °C for 3 min; 6 cycles at 94 °C for 45 s, 55–65 °C for 1 min, and 72 °C for 1 min; 9 cycles of 94 °C for 45 s, 50–58 °C for 1 min, and 72 °C for 1 min; 19 cycles at 94 °C for 30 s, 50 °C for 30 s, and 72 °C for 1 min; and final extension at 72 °C for 5 min. PCR products were analyzed by electrophoresis on 8.0% (w/v) denaturing polyacrylamide gel in TBE buffer for 1 h on the DYY-6 C electrophoresis apparatus (Beijing Liuyi Instrument Factory, China) at a constant voltage of 220 V. DNA fragments were visualized by silver staining (Silver sequence staining reagents, Promega, Madison, USA) and sized with a 50 bp DNA ladder marker (Tiangen, China) [47 (link)]. SSR primer sequences are listed in Supplementary Table S2.

Transfected cells were sorted for RNA extraction to assess the A-to-I base-editing efficiency of mini-Vx and Vx. A FastKing RT kit (with gDNase) (TIANGEN Biotech, China) was used to reverse transcribe complementary cDNAs from RNAs, and crRNA target sites were amplified from cDNAs using 2*Taq PCR Master Mix II (TIANGEN Biotech, China) for Sanger or deep sequencing. The Sanger sequencing results were analyzed with EditR to measure the effectiveness of the A-to-I conversion at each target site (http://baseeditr.com/)^{54 (link)}. Deep sequencing was performed by using Hi-TOM (http://www.hi-tom.net/hi-tom/index-CH.php)^{55 (link)}. All Cas13 crRNAs used in this work are listed in Tables S4 and S5. The primers are shown in Table S2.

Total RNA was extracted at the indicated time points (4, 8, 12, 24, 36, and 48 hpi) from cells using TRNzol Universal Reagent (Tiangen, Beijing, China). RNA was reverse transcribed using Fastking gDNA Dispelling RT SuperMix (Tiangen, Beijing, China), and PCR was performed with 2 × Taq PCR MasterMix II (Tiangen, Beijing, China) using the primers listed in Supplemental Table S1. PCR reactions were 94 °C for 3 min, 35 cycles of 94 °C for 30 s, 58 °C for 30 s, and 72 °C for 20 s, followed by 72 °C for 5 min. Amplicons were separated on 2% agarose gel and examined using the Alphaimager EC imaging system (Alpha Innotech, San Jose, CA, USA).

For uniplex PCR, the 20 μL mixture containing 2 μL cDNA template,10 μL 2× Taq PCR MasterMix II (Tiangen, Beijing, China), 0.5 μL each of forward/reverse primers (10 μM), and 7 μL ddH₂O. For multiplex PCR, the 20 μL mixture containing 2 μL cDNA template, 10 μL 2× Taq PCR MasterMix II, 4 μL of multiplex primer mixture (containing 0.5 µL each of forward and reverse primer for OMSV, OMIV, POSV, and PoV1, 10 μM), and 4 μL ddH₂O. A multiplex PCR program was set as follows: pre-denaturation at 95 °C for 5 min, followed by 30 cycles of 3 steps including 95 °C for 30 s, 62 °C for 30 s, followed by 72 °C for 60 s and a final extension at 72 °C for 10 min. After PCR amplification, the products were examined by electrophoresis in 1.5% agarose gel. The healthy P. ostreatus sample (virus-cured strain 8129 [31 (link)]) acted as a negative control.

FPKM values of 41 SmTCPs were screened from RNA-seq data to analyze the expression of SmTCP genes. The gplots [39 ] and pheatmap [40 ] packages were used to draw the heat map (FPKM values).
We screened for the differentially expressed TCPs (DETCPs) from all DEGs (differentially expressed genes) under B1–R1, B2–R2, and B3–R3, defined as |log2FC|>1 and FDR ≤ 0.05. DETCPs were further analyzed.
We designed gene-specific qRT-PCR primers for DETCPs. The plant materials used for the RT-PCR, to verify the RNA-seq results, were identical to those used for the RNA-seq. RNA was extracted from the bud base of cutting seedlings of S. muricatum treated with light of different qualities, with B, R and F light treatment for 10 d, 20 d, and 30 d, and six DETCPs in three subclusters (CYC/TBI, CIN, and Class 1) were randomly selected with reference to expression profiling. The detailed parameters of each primer pair are given in Table S1. We followed a two-step qRT-PCR protocol for cDNA synthesis (FastKing gRNA Dispelling RT SuperMix, KR118, TIANGEN BIOTECH(BEIJING)CO., LTD) and amplification in successive steps (2*Taq PCR MasterMix II, KT211, TIANGEN BIOTECH CO., LTD) to reduce the undesired primer dimer formation using SYBR Green. The RT-PCR data were analyzed by the 2^-ΔΔCt method.