Primerscript rt kit

To corroborate the RNA-Seq results, we randomly selected 10 genes from the SDEGs for qRT-PCR validation, including five upregulated genes and five down-regulated genes. Chicken spleen tissue total RNA was extracted, and a reverse transcription reaction was carried out using a primerScriptTM RT kit (Takara, Kyoto, Japan) to obtain cDNA for qRT-PCR. The reaction was carried out using the LightCycler^® 96 instrument qRT-PCR system (Roche, Basel, Switzerland) and SYBR^® PremixEx TaqTM kit (Takara, Kyoto, Japan). The qRT-PCR amplification procedure was as follows—95 °C for 3 min, 35 cycles of 95 °C for 30 s, 60 °C for 30 s, 72 °C for 30 s, and extended at 72 °C for 10 min. In addition, before the gene quantification, three common chicken housekeeping genes (B2M, β-actin and GAPDH) were tested [16 (link)] and evaluated using GeNorm [17 (link)]. Since GAPDH showed a lower M value and was stable in Gushi chickens [18 (link)], it was used as an internal reference gene. The relative expression changes of the genes were calculated using the 2^−ΔΔCt method. The qRT-PCR samples were the same as the RNA-seq samples, with three biological repeats for each group and three repetitions for each sample. The primer sequences are listed in (Additional File 3: Table S2).

Total RNA was extracted from cells, and synthesis of the first cDNA strand was performed by using a PrimerScript TM RT kit (Takara, Dalian, China). The primer pairs for the genes encoding Bax, Bcl-2, caspase-3, caspase-8, Fas, p53, and glyceraldehyde-3-phosphate dehydrogenase (a housekeeping protein) were designed by using Premier 5.0 software (Premier Biosoft, Palo Alto, CA, USA) and synthesized by Shanghai Biological Technology Co., Ltd. (Shanghai, China). The PCR conditions were as follows: denaturation at 94 °C for 5 min, 30 cycles of 30 s each at 94 °C, 30 s of annealing, 72 °C for 30 s and a 72 °C extension for 10 min. PCR products were identified via electrophoreses on 1.5 % agarose gels.

Total RNA was extracted from the tepals of I. laevigata using a kit (Kangweishiji, Taizhou, China), and the integrity was checked by electrophoresis. Frist-strand cDNA was synthesized using the Primer Script TM RT kit (TakaRa, Beijing, China). IlWRKY22 (accession number: ON399552) was cloned using KOD-plus-neo (ToYoBo, Shanghai, China) with primers (Ruibo Kexing, Harbin, China) listed in Table S1. The cloned sequences were inserted into the Cloning vector pEASY^®-Blunt Zero Cloning Kit (TransGen, Beijing, China) and transformed into Escherichia coli DH5α (WEIDI, Shanghai, China). The pCAMBIA1300: IlWRKY22-GFP carrier was constructed for heterologous expression through homologous recombination. Homologous recombination primers (Ruibo Kexing, Harbin, China) listed in Table S1. The pCAMBIA1300 vector were digested with SalI and BamHI, which were then used to create pCAMBIA1300-IlWRKY22-GFP using ClonExpressII (Vazyme, Nanjing, China). The recombinant vector was transformed into E. coli DH5α (WEIDI, Shanghai, China) for sequencing. Positive plasmids were transferred into Agrobacterium tumefaciens GV3101 (WEIDI, Shanghai, China) for subsequent infection experiments. Bioinformatics analysis of IlWRKY22 was performed as described [26 (link)].

Total RNA was isolated from homogenized liver tissues using a TRIzol™ isolation kit (Takara Bio, Dalian, China) following the manufacturer's protocol. The cDNA was synthesized by using Primer Script RT kit (Takara Bio, Dalian, China). Prime Script™ RT reagent kits, along with SYBR Green Realtime PCR Master Mix and Permix Ex Taq (Takara Bio), according to the manufacturer's instructions. The primers for GAPDH, TNF-α, IL-10, TGF-β1, α-SMA, and Desmin were synthesized by Sangon Biotech (Shanghai, China). Real-Time PCR was operated on ABI Prism 7500 Sequence Detection System (BioRad, Life Science Research, Hercules, CA, USA). PCR conditions were as follows: one cycle at 95°C for 30 s, 40 cycles at 95°C for 5 s, at 64°C for 30 min. All reactions were performed in triplicate for each sample. The 2^−ΔΔCT method was used to calculate relative concentration of each target by standardizing to internal GAPDH level.

Oxidative stress is a biochemical reaction and leads to a series of biological effects and cell damage [21 ,22 (link),23 (link)]. We selected ten genes related to oxidative stress and the gapdh gene as the internal reference gene. We extracted the total RNA of 30 zebrafish larvae from each group using RNAisoPlus reagent (Takara Bio Inc., Shiga, Japan) according to the manufacturer’s instructions. The quality of total RNA was measured using a spectrophotometer and 1% agarose gel electrophoresis. Next, we synthesized cDNA using the Primer Script RT kit (Takara Bio, Shiga, Japan) with 1 μg RNA of each sample following the manufacturer’s instructions. qRT-PCR was performed using the ABI 7300 system (PerkinElmer Applied Biosystems, Shelton, CT, USA) with the TB Green PCR kit (Toyobo, Tokyo, Japan). The primers of zebrafish were designed using the NCBI Primer online tool (The primer sequences can be seen in the SI). The PCR program was as follows: 95 °C for 30 s, 95 °C for 20 s, 58 °C for 20 s, and 72 °C for 45 s for 40 cycles. At the end of the amplification process, we achieved the dissociation curve with 95 °C for 15 s, 60 °C for 1 min, and 95 °C for 15 s. We performed three replicates of all groups and used the 2^−ΔΔCT method to quantify the target gene expression levels.

Total RNA from SH-SY5Y cells and BV2 cells was isolated with a commercial kit (Rneasy Mini kit, Qiagen, Valencia, CA, USA) after collecting samples in TRIzol reagent (Thermo Fisher Scientific, Madrid, Spain) according to the manufacturer’s instructions. RNA concentration and purity were assessed by OD measurements at 260 nm and 280 nm on a NanoDrop spectrophotometer. The integrity of RNA was confirmed in 1% agarose gels after electrophoresis. cDNA synthesis was performed using 200 ng of RNA and the Primer script RT Kit (Takara, Madrid, Spain) following the manufacturer’s instructions.

To verify the accuracy of the obtained differential gene expression patterns between fiber and nonfiber tissues, we screened 36 genes that were significantly upregulated in fibers for verification analysis by qRT-PCR. These DEG-specific primers were designed with the Primer3 online tool (http://bioinfo.ut.ee/primer3-0.4.0/) (Table S5). According to the manufacturer's instructions, cDNA synthesis was performed from 1 μg of total RNA in a 20 μL reaction mixture using a PrimerScript RT kit (TAKARA, Dalian, China). The 20 μL reactions were performed using 10 μL of SYBR Premix Ex Taq II (TLi RanseH Plus) (TAKARA, Dalian, China), 0.8 μL of 10 mM forward and reverse primers each, 7.4 µL of ddH₂O and 1 μL of cDNA template, after which amplification reactions were conducted. The cotton Sad1 gene was used as an internal reference gene. The qRT-PCR conditions were as follows: 95 °C for 30 s, 40 cycles of 95 °C for 5 s and 60 °C for 34 s. Three biological and technical replicates were performed for each sample to verify the results of the qRT-PCR test, and relative gene expression levels were quantified via the 2-ΔΔCt method.