2xtaq plus master mix dye plus

To construct a stable PD-L1-3′-UTR knockout cell line, SUM-159 cells were co-transfected with a combination of designed sgRNAs and transfected by the epiCRISPR vector plasmid as the control using Lipo2000 (Invitrogen, Waltham, MA, USA). After 48 h of transfection, the medium was replaced by complete medium containing 2 μg/mL puromycin (InvivoGen, San Diego, CA, USA) for 7 days to select stable cell lines. The stable cell line was confirmed by genotyping. The cells were lysed by 50 μL 1x mouse tissue lysis buffer (Vazyme) with 1 μL protease K (Vazyme) at 55 °C for 30 min. Then, the genomic PCR was performed with2xTaq Plus Master Mix (Dye Plus) (Vazyme) under the manufacturer’s instructions. The PCR products were analyzed with agarose gel electrophoresis. The DNA was purified for DNA sequencing, and the DNA sequencing was performed by Suzhou Anshengda Company, Suzhou, China.

First-strand cDNAs were synthesized using the Prime Script™ RT Reagent Kit with gDNA Eraser (Takara, Kyoto, Japan). Semi-quantitative PCR reactions were performed using 2xTaq Plus Master Mix (Dye Plus) (Vazyme, Nanjing, China). The PCR conditions were as described previously [6 (link)]. Three biological repetitions were performed. The specific primers for the selected genes and the internal control (Gossypium hirsutum polyubiquitin protein gene, Gbp) are listed in Table S1.