Phanta max master mix dye plus

To obtain the genome template, the single colony was dispersed in 20 μL of a 0.25% SDS solution, followed by incubation at 90 °C for 3 min. The PCR was performed using the 2× Phanta Max Master Mix (Dye Plus) from Vazyme Biotech Co., Ltd. (Nanjing, China). And the resulting products were verified by DNA electrophoresis or sequenced as necessary by Sangon Biotech Co., Ltd. (Shanghai, China). eSpCas9 was confirmed using primer pairs eCAS9-F/eCAS9-R; TRP1 was confirmed using primer pairs TRP-F/TRP-R; and LIP2 was confirmed using primer pairs LIP-F/LIP-R.

C. saxicola was identified by Drs Zhan-jiang Zhang and Cui Li of the Guangxi Botanical Garden of Medicinal Plants (GBGMP) (Nanning, Guangxi Zhuang Autonomous Region, China). Voucher specimens (reference number: 450123130505040LY) were deposited in the GBGMP Herbarium.
Fresh and healthy C. saxicola tissues were collected from the greenhouse located in the GBGMP experimental area. One gram of C. saxicola leaves were used four total DNA extraction according to the 2 × cetyltrimethylammonium bromide method (Porebski et al., 1997 (link)). The same qualified DNA sample was used for Illumina sequencing, Oxford Nanopore sequencing, and PCR amplification.
Thirty mg of C. saxicola roots, stems, branches, mature leaves and young leaves were used for total RNA extraction according to the manual of RNA Isolater Total RNA Extraction Reagent (Vazyme, China). The first-strand cDNA was synthesized using a HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper) (Vazyme, China). Qualified extracted RNA was used as a template, and random hexamers and oligo(dT)₂₀VN were used as primers. PCR was performed using 2 × Phanta Max Master Mix (Dye Plus) (Vazyme, China) and conducted on a T100 Thermal Cycler (BIO-RAD, USA).

The cells were washed with PBS buffer three times and then incubated with 1 mL Trizol lysis buffer for 10 min. The cell lysates were collected and further incubated with 0.2 mL chloroform for 3 min, followed by centrifugation at 12000 g, 4 °C for 15 min. The upper colorless aqueous phase was carefully transferred into a new 1.5 mL EP tube, gently mixed with an equal volume of isopropyl alcohol, and stood at room temperature for 10 min. After centrifugation at 12000 g, 4 °C for 10 min, the supernatant was discarded. The pellet was washed with ice‐cold 75% ethanol and dissolved in an appropriate amount of DEPC (RNase Free) water. The concentrations and purities of all RNA samples were determined, and the reverse transcription was performed to synthesize cDNA using HiScript 1st Strand cDNA Synthesis Kit (Vazyme). Gene expression levels were determined using 2 × Phanta Max Master Mix (Dye Plus) (Vazyme).

Two micrograms of total RNA was subjected to first-strand cDNA synthesis using a TransScript First-Strand cDNA Synthesis SuperMix (TransGen Biotech, Cat# AT301-02, Beijing, China) and an oligo (dT18) primer. AS was validated by RT-PCR using 2 × Phanta Max Master Mix (Dye Plus) (Vazyme, Cat# P525-01, Nanjing, China) with 5 × diluted cDNA as the template. The PCR products were visualized in 1.2% agarose gel stained by GelStain. The APA events were validated using a 3′ RACE Kit (Clontech, cat # 6106) according to the manufacturer’s instructions. Primer lists used in the validation are listed in Supplementary Table S1.

Total RNA was extracted from pig MuSCs. PCR primers flanking pig YAP1 were designed based on the published predicted sequence in NCBI database (Accession#: XM_021062706). PCR experiments were performed with 2 × Phanta^® Max Master Mix (Dye Plus) (Vazyme, Nanjing, China, Cat# P525-01) following the manufacturer’s protocol. We then cloned the resultant YAP1 CDS into the pEASY^®-Blunt Simple Cloning Vector (Transgen, Nanjing, China, Cat# CB11) and verified the sequence via Sanger sequencing. The YAP1 CDS sequences and protein sequences were aligned by Clustal Omega (https://www.ebi.ac.uk/Tools/msa/clustalo/, 29 October 2021) supported by The European Bioinformatics Institute (EBL). The primers used were (accggt indicate restriction site for AgeI): F: 5′-accggtATGGATCCCGGGCAGCAGCAGCCGC-3′, R: 5′-accggtCTATAACCATGTAAGAAAGCTTTC-3′.

All the DNA fragments were amplified using 2 × Phanta Max Master Mix (Dye Plus) (Vazyme, Nanjing, China). A ready-to-use seamless cloning kit (Sangon, Shanghai, China) was used for plasmid construction; the length of the homologous arm was not less than 25 bp between the fragments. All the recombinant plasmids were determined by DNA sequencing. The knockout and integration of genes were performed using CRISPR-Cas9 systems (Dicarlo et al., 2013 (link)). The CRISPRdirect tool (http://crispr.dbcls.jp) was used for selecting a 20-nt guide sequence of the single-guide RNA (sgRNA). All the 20-nt guide sequences used in this study are listed in Supplementary Table S3. The gene deletion and expression cassettes (donor DNA) for genome editing were obtained by overlap extension polymerase chain reaction, in which the homologous arms were amplified from the genome and were not less than 300 bp. A Frozen-EZ Yeast Transformation II Kit (Zymo Research, CA, United States) was used for the transformation of S. cerevisiae following the manufacturer’s protocol.