Reverse transcriptase kit

Total RNA was extracted from tissues and EVs as described previously. In brief, total RNA was extracted from all samples by RNA isolater Total RNA Extraction Reagent (Vazyme, China), and cDNA was synthesized using a reverse transcriptase kit (Vazyme, China). For tissue samples from the DSS group, the isolated RNA was purified using LiCl. In brief, a 0.1 volume of 8 M LiCl (Sigma-Aldrich) solution was added to a 1 volume of RNA solution and incubated on ice for 2 h before being centrifuged (14000 × g, 30 min, 4°C), after which the pellets were collected, and the process was repeated twice. Subsequently, 0.1 volume of 3 M sodium acetate (Thermo Scientific) and 2 volumes of ethanol were added to 1 volume of RNA solution to reprecipitate RNA without DSS. For EV samples, a settling agent (Takara #9094, Japan) was added with ethanol to extract the miRNAs. Subsequently, before reverse transcription, the RNA was processed with Poly(A) polymerase (NEBio, China) for the tailing reaction. The primers for miRNA reverse transcription were designed by miRprimer.^{64 (link)} Quantitative real-time PCR (RT-PCR) was performed as described previously.^{65 (link)} The primer sequences are shown in Table S1.

TRIzol (Thermo Fisher, Cat# 15596026) was used to extract total RNA from BMMs. The reverse transcriptase kit (Vazyme, Cat# R223) was used to reverse transcribe an aliquot of 200 ng total RNA into cDNA. SYBR green mixture (Vazyme, Cat# Q311) and Monad Real-Time PCR instrument (Monad q225) were used for quantitative PCR. The primers used for specific transcripts are listed in Additional file 8: Table S1. The mRNA level was normalized to that of GAPDH.

The total RNA from buffalo SCs was extracted using the RNAiso Plus kit (Takara, Beijing, China). The quality of the total RNA was evaluated by a microplate spectrophotometer (Epoch, BioTek, VT, USA). Reverse transcription of the total RNA (≈1.5 μg) to cDNA was done using a reverse transcriptase kit (Vazyme, Nanjing, China). Real-time quantitative PCR (qRT-PCR) analysis was performed to amplify the target genes using SYBR qPCR Master Mix (Vazyme, Nanjing, China), and the reaction was conducted on a CFX96 Touch Real-Time PCR Dection System (Bio-Rad, CA, USA). Reactions were performed in a final volume of 20 µL with 0.4 µL of each primer (≈4 µM), 2 µL of cDNA (≈150 ng), 10 µL of SYBR qPCR Master Mix, and 7.2 µL of ddH₂O. The reaction was run with the following parameters: 95 °C, 30 s, followed by 95 °C, 10 s and 60 °C, 30 s for 40 cycles. Melting curves were acquired using the default parameters of the CFX96 Touch Real-Time PCR Detection System. Negative qRT-PCR controls were executed by ddH₂O and RNA, instead of cDNA. Every reaction was repeated three times and were normalized against GAPDH expression [53 (link),54 (link),55 (link),56 (link)]. The 2^−∆∆CT approach [57 (link)] was used to calculate the relative expression levels of the chosen BRD genes in buffalo. Table S1 lists the primers used in the qRT-PCR experiments.

TRIzol Reagent (Vazyme, Nanjing, China) was used to extract total RNA. 1 μg RNA was reverse transcribed into cDNA using reverse transcriptase kit (Vazyme). Quantitative real-time PCR assays were performed using ABI7500 (Applied Biosystems) in 8 strips caps (Axygen, Corning, USA) with the SYBR Green PCR Master Mix (Rox, Vazyme). The fold changes were calculated using the 2^−△△Ct method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a reference gene. All the primer sequences were presented as follows: GAPDH forward 5′-GTCATCCCTGAGCTGAACGG-3′, reverse 5′-AAGTGGTCGTTGAGGGCAAT-3′; CTNNB1 forward 5′- CACAAGCAGAGTGCTGAAGGTG-3′, reverse 5′- GATTCCTGAGAGTCCAAAGACAG-3′; CCND1 forward 5′-GATGCCAACCTCCTCAACGA-3′, reverse 5′-GGAAGCGGTCCAGGTAGTTC-3′; MYC forward 5′-CGTCCTCGGATTCTCTGCTC-3′, reverse 5′-GCTGCGTAGTTGTGCTGATG-3′.

TRIzol Reagent (Vazyme, #R401-01) was utilized to extract total RNA according to the manufacturer’s instructions, and concentrations of RNA were determined by NanoDrop 2000 (Thermo Fisher). 1 μg of RNA was reverse-transcribed into cDNA using a reverse transcriptase kit (Vazyme, #R211-01). Quantitative real-time PCR assays were performed using ABI7500 (Applied Biosystems) in 8 strips caps (Axygen, Corning, NY, USA) with SYBR Green PCR Master Mix (Vazyme, #Q331-02). The fold-changes were calculated using the 2^—△△Ct method, with glyceraldehyde-3-phosphate dehydrogenase (GAPDH) serving as a reference gene. All the primer sequences were presented in Table S2.