Nova 6000 platform

Nine male samples (including 5 BA.2-BTI-6m and 4 controls) were used for single-cell sequencing. The single-cell suspension was prepared in water for cDNA library amplification using the 10× Genomics Chromium Next GEM Single Cell 5ʹ Reagent Kits (version 2.0; Cat. No. 1000165). The Chromium™ Single Cell 5ʹ Library Construction Kit (Cat. No. 1000020) was used to construct the DNA library. The constructed library was then sequenced using PE150 sequencing on an Illumina Nova 6000 platform. T cell V(D)J and B cell V(D)J enrichment analyses were performed using the 10× Genomics Chromium™ Single Cell V(D)J Enrichment Kit, Human T Cell (Cat. No. 1000005) and Human B Cell (Cat. No. 1000016) according to the manufacturer’s instructions. The libraries were amplified using a Chromium TCR amplification kit (Cat. No. 1000252) and BCR amplification kit (Cat. No. 1000253), and the experiment was performed according to the manufacturer’s instructions.

DNA Isolation Kit, which is produced by MP Biomedicals in Switzerland, was used to extract DNA from 0.5 g of fresh soil samples. The extraction process followed the manufacturer’s recommendations. The extracted DNA was assessed for its quality and quantity using a Nanodrop ND-2000 UV-vis spectrophotometer produced by Nanodrop Technologies in Wilmington, DE, USA. Primers 515F (5′-GTGCCAGCMGCCGCGGTAA-3′) and 806R (5′-GGACTACHVGGGTWTCTAAT-3′) were used to enhance the bacterial 16S rRNA gene V4 hypervariable region (Dong et al., 2022 (link)). PCR reactions were performed in triplicate (S1000 apparatus, Bio-Rad Laboratory, Hercules, CA, USA). The PCR conditions were 5 min at 94°C followed by 30 cycles of 94°C for 45 s, annealing at 54°C for 45 s, 72°C for 1 min followed by a final extension step of 10 min at 72°C (Katiraei et al., 2022 (link)). PCR products were purified using the Qiagen Gel Extraction Kit produced by Qiagen in Germany, following the instructions provided by the manufacturer. In the end, the library was sequenced using an Illumina Nova6000 platform, which produced paired end reads of 250 base pairs. The sequencing procedure was conducted by Guangdong Magigene Biotechnology Co., Ltd., situated in Guangzhou, China.

We obtained ear tissue from 20 unrelated Altay white-headed cattle from the core breeding tracts of Altay, Xinjiang Province, China. Genomic DNA was extracted from the samples using a standard phenol–chloroform protocol (Toni et al., 2018 (link)). Paired-end libraries were constructed for each individual (350 bp insert size) and sequenced, using the Illumina-Nova 6000 Platform with a 2 × 150 bp model. Additionally, we used 144 publicly available genomes from NCBI, including Bohai cattle (n = 9), Brahman cattle (n = 4), Charolais cattle (n = 13), Hereford cattle (n = 13), Holstein cattle (n = 22), Kazakh cattle (n = 9), Mongolian cattle (n = 17), Simmental cattle (n = 16), Wannan cattle (n = 5) and Yanbian cattle (n = 16) (Supplementary Table S1).

Gene-specific DNA methylation was assessed by a next generation sequencing-based targeted bisulfite sequencing, according to previously published method [18 (link)–21 (link)]. In brief, primers were designed using the online MethPrimer software and listed in Table 2. 200 ng of genomic DNA was converted using the ZYMO EZ DNA Methylation-Gold Kit (Zymo Research, Irvine, CA, USA) and were used as templates for PCR amplification with 35 cycles using KAPA HiFi HotStart Uracil+ ReadyMix PCR Kit (Kapa Biosystems, Wilmington, MA, USA). For each sample, PCR products of multiple genes were pooled equally, 5’-phosphorylated, 3’-dA-tailed and ligated to barcoded adapter using T4 DNA ligase (NEB). Barcoded libraries from all samples were sequenced on Illumina Nova6000 platform using a 150×2 paired-end sequencing protocol.

Fecal DNA was extracted using QIAamp PowerFecal Pro DNA kit (Catalog no. 51804; Qiagen, Inc., Germany) following the manufacturer’s protocol. The quantity and quality of DNA were measured using a Biodropsis BD-1000 spectrophotometer (OSTC, Inc., China) and agarose gel electrophoresis, respectively. PCR amplification of the bacterial 16S rRNA gene V4 region (F515/R806) was performed according to previously described protocols (47 (link)). All PCR amplicons were mixed and sequenced using the Illumina Nova 6000 platform (2 × 250 bp, paired-end) following the manufacturers protocol. All DNA samples from 70 fecal samples were subjected to the same sequencing run.