Primescripttm rt master mix reverse transcription kit

For the epidemiological survey of PSV, a total of 260 clinical samples, including feces, fecal swabs, and intestine samples, were collected between 2020 and 2022 from sows and suckling, post-weaning, and fattening pigs that were asymptomatic or with clinical diarrhea (25 with clinical diarrhea and 5 asymptomatic) from 30 farms in Fujian Province. A description of the studied pig population and the number of collected samples for each farm is shown in Supplementary Table S1. All samples were detected for PSV using real-time PCR. The PSV2020 strain was preserved by the Institute of Animal Husbandry and Veterinary Medicine, Fujian Academy of Agricultural Sciences. M-MuLV First Strand cDNA Synthesis Kit (Code No: B532435), Rapid Competent Cell Preps Kit (One-step, Code No: B529307), SanPrep Column Plasmid Mini-Preps Kit (Code No: B518191), SanPrep Spin Column & Collection Kit (Code No: B515103), and Taq Plus DNA polymerase, 2 × Taqman Fast qPCR Mix (High Rox) (Code No: B639276, BBI) were bought from Sangon Biotech (Shanghai, China). RNA extraction kit (Code No: 9767), PrimeScriptTM RT Master Mix Reverse Transcription Kit (Code No: RR036A), TaKaRa LA Taq^® (Code No: RR02MQ), and pMD 18-T Vector (Code No: 6011) were purchased from TaKaRa Bio Inc. (Daliang, China).

F-12 K medium (BOSTER, USA), fetal bovine serum (Life Technology 10,099,141, USA), horse serum, DMSO (Solebao, China), penicillin/streptomycin (Yuanpei, China), trypan blue dye Liquid (Melen, China), trypsin-EDTA, BCA protein content determination kit (Jin Yibai, China), poly-L-lysine (source leaf, China), nerve growth factor (Boaosen, China), NSE, MAP 2 antibody (abcam, USA), Grp78, CHOP antibody (Santa Cruz, USA), secondary antibody (Bio-Rad, USA), thapsigargin (TG), icariin (crystal pure, China), CCK8 kit (Biosharp, China), Sulfa Rhodamine B (SRB) Kit (Beibo, China), Annexin V-FITC Apoptosis Kit (Biyuntian, China), TaKaRa PrimeScriptTM RT Master Mix Reverse Transcription Kit, TaKaRa TB GreenTM Premix Ex Taq TMII PCR kit (TaKaRa, Japan) .

To validate the RNA-seq results, 10 structural genes and 4 transcription factors (TFs) involved in the anthocyanin biosynthesis were subjected to qRT-PCR using gene-specific primers (Table S10). These gene-specific primers were designed using Primer 3 software according to the gene sequence. The expression level of the target gene was determined with the EF-1-α gene (GenBank login number: GO479260) as an internal reference. The total RNA of each sample was extracted at b1, b2, b3, b4, and w4 according to the TRIzol Reagent User Guide (Life Technologies, Carlsbad, CA, USA). The first-strand cDNA was obtained using the PrimeScript^TM RT Master Mixreverse transcription Kit (Takara, Dalian, China). QRT-PCR was repeated three times in a 20 μL reaction system containing 10 μL of 2 × TB Green Premix EX Taq, 2.0 μL of diluted cDNAs, 0.4 μL of 50 × ROX Reference Dye II, 0.4 μL of forward primers (10 μM), 0.4 μL of reverse primers (10 μM), and sterile ddH₂O. PCR was carried out in an ABI 7500 instrument (ABI, Oakland, CA, USA), and the thermal cycling parameters were as follows: 30 s at 94 °C, followed by 40 cycles (30 s at 95 °C, 30 s at 60 °C, and 30 s at 72 °C). The relative gene expression level was calculated using the 2^−ΔΔCT method [44 (link)].