Hiseq ten platform

The complete genome of strain SCA2-4^T was sequenced using a paired-end sequencing method in the Illumina Hiseq × Ten platform (Illumina, San Diego, CA, United States) by the Shanghai Majorbio Bio-pharm Technology Co., Ltd., (Shanghai, China). Data were analyzed on the free online platform of the Majorbio Cloud Platform³. Clean sequencing data were optimized using the GapCloser v1.12 (Luo et al., 2012 (link)) and were subsequently assembled using the SOAPdenovo v2.04. The protein-coding genes were predicted by the Glimmer v3.02 (Delcher et al., 2007 (link)⁴). Gene functions were annotated using six databases (NR, Swiss-Prot, Pfam, EggNOG, GO, and KEGG). The biosynthetic gene clusters of secondary metabolites were predicted using the online antiSMASH v 6.0.1 software (Kai et al., 2017 (link)⁵).

The Illumina library for each tissue sample was constructed using the TruSeq RNA Sample Prep Kit (Illumina) following the manufacturer’s instructions. Briefly, the polyA mRNA was fragmented using divalent cations at elevated temperature. The RNA fragments were reverse transcribed into first-strand cDNA using reverse transcriptase and random primers, followed by second-strand cDNA synthesis, end repair, and ligation of the adapters. The ligated fragments were purified and enriched through PCR to generate the final cDNA library. Finally, 12 transcriptomic libraries (six libraries from each group) were sequenced on Illumina HiSeq × Ten platform to obtain 150 bp pair-end reads. Raw RNA-seq reads in fastq format were first filtered through in-house perl scripts to filter out the low-quality reads. Reads with a Q30 percentage greater than 85% were retained as high-quality reads; the rest of the reads as low-quality reads were filtered out. Then clean reads were obtained by removing reads containing sequencing adapters, ploy-N, and low quality. The clean paired-end reads from each library were merged together and then de novo assembled by using Trinity 2.8.4 software with the default parameters. The clean short reads were then mapped to the PacBio reference sequence using Tophat2 tools.

All the experimental conditions of this study were approved by the Committee on the Ethics of Animal Experiments of the Southwest University (No. [2007] 3) and the Animal Protection Law of China.
We collected venous blood samples from 55 goats comprising 35 intersex goats (26 XX Tangshan dairy goats and 9 XX Chinese southern native goats) and 20 XX non-intersex Tangshan dairy goats. A total of 2 mL venous blood was collected from each animal (Sampling from Tangshan dairy goat breeding farm, Tangshan, China). The wound was sterilized with 70% medical alcohol. All 55 animals were returned to the pasture to continue living after experimentation. All genomic DNA samples were extracted by using a QIAGEN DNeasy Blood & Tissue kit in accordance with the manufacturer's protocol. Sequencing libraries were constructed with DNA extracts and a NEBNext® Ltra DNA library preparation kit (Illumina®, US). Sequencing was performed on an Illumina HiSeq × Ten platform (pair-end 150 bp). The sequencing data generated in this study were deposited in the NCBI SRA database (SRR10051499-SRR10551533 and SRR10613872-SRR10613891). In addition, we downloaded 166 non-intersex goat genome sequences from the NCBI SRA database. The detailed information of the 221 animals used in this study is shown in Supplementary Table 1.

Total RNA was extracted using TRIzol reagent (Invitrogen, Carlsbad, CA, USA) according the manufacturer’s instructions. RNA concentration was determined using a NanoDrop-2000 spectrophotometer (Thermo Scientific, Waltham, MA, USA) and RNA integrity was determined by agarose gel electrophoresis. Poly(A) mRNA was isolated from the total RNA using Oligo (dT) and cleaved into short fragments using a Covaris S220 Focused-Ultra sonicator (Covaris, California, USA) and then used as templates for the synthesis of first- and second-strand cDNA according to the protocol of the Super Script Double-Stranded cDNA Synthesis Kit (Thermo Fisher Scientific, MA, USA). The cDNA was purified using a QIAquick PCR Purification Kit (QIAgen, Düsseldorf, Germany). After end repair, poly(A) addition and sequencing adapter ligation, the optional 300–400 bp fragments were selected by agarose gel electrophoresis, and enriched by PCR amplification to construct the cDNA library, followed by sequencing and generation of 150 bp paired-end reads using the Illumina HiSeq Ten platform. Three independent biological replicates were performed for stage Ⅱ and Ⅳ based on each tissue.

Strain 8ZJF-21 was cultured in the ISP2 liquid medium at 200 rpm and 28°C for 4 days. Total genomic DNA was extracted using a Rapid Bacterial Genomic DNA Isolation Kit (Biotake corporation, Beijing, China). The sequencing libraries were generated using the Illumina TruSeq™ RNA Sample Preparation Kit (Illumina, San Diego, CA, United States). The complete genome was sequenced in the Illumina Hiseq × Ten platform (Illumina, San Diego, CA, United States) by the Shanghai Majorbio Bio-pharm Technology Co. Ltd. Sequencing data were analyzed using an online platform of the Majorbio Cloud¹ and was deposited in GenBank with accession number JAJQWY000000000. The open reading frames (ORFs) were predicted by the Rapid Annotation using Subsystem Technology (Brettin et al., 2015 (link)). Functional annotation was performed using the Clusters of Orthologous Group (COG), the Gene Ontology (GO), and the Kyoto Encyclopedia of Genes and Genomes (KEGG) (Ogata et al., 1999 (link); Tatusov et al., 2000 (link)). Biosynthetic gene clusters (BGCs) were identified by the online antiSMASH v4.0.2 software (Weber et al., 2015 (link)).