Novaseq 6000 machine

DNA libraries were prepared for 1261 D. sylvestris individuals from 115 populations (5–20 individuals per population) under a modified protocol⁴⁹ of the Illumina Nextera DNA library preparation kit (Supplementary Methods S1.1, Supplementary Data 1). Individuals were indexed with unique dual-indexes (IDT Illumina Nextera 10nt UDI – 384 set) from Integrated DNA Technologies Co, to avoid index-hopping^{50 (link)}. Libraries were sequenced (150 bp paired-end sequencing) in four lanes of an Illumina NovaSeq 6000 machine at Novogene Co. This resulted in an average coverage of ca. 2x per individual. Sequenced individuals were trimmed for adapter sequences (Trimmomatic version 0.35^{51 (link)}), mapped (BWA-MEM version 0.7.17^{52 (link),53} ) against a reference assembly⁵⁴ (ca. 440 Mb), had duplicates marked and removed (Picard Toolkit version 2.0.1; http://broadinstitute.github.io/picard), locally realigned around indels (GATK version 3.5⁵⁵ ), recalibrated for base quality scores (ATLAS version 0.9⁵⁶ ) and had overlapping read pairs clipped (bamUtil version 1.0.14^{57 (link)}) (Supplementary Methods S1.1). Population genetic analyses were performed on the resultant BAM files via genotype likelihoods (ANGSD version 0.933^{58 (link)} and ATLAS versions 0.9–1.0⁵⁶ ), to accommodate the propagation of uncertainty from the raw sequence data to population genetic inference.

Blood drawn during the OPTIMISTIC trial was collected in Tempus tubes and centrally stored at the New Castle MRC Centre for Rare & Neuromuscular Diseases biobank with strict SOPs and temperature control (−80°C). RNA was locally isolated in Nijmegen using the Tempus Spin RNA Isolation Kit (Applied Biosystems/Thermo Fisher Scientific) according to the manufacturer’s instructions. The concentration and RNA Integrity Number (RIN) were checked using Fragment Analyzer (Thermo Fisher Scientific). The mean RIN value was 8.9 and all were > 7.5. Hemoglobin mRNA was depleted using the Globinclear kit (Thermo Fisher Scientific). Libraries were prepared using NEBNext Ultra II Directional RNA Library Prep Kit (Illumina) according to the manufacturer’s instructions for a polyA mRNA workflow using UMI-indexed adapters. The size distribution (between 300 and 500 bp) was confirmed using Fragment Analyzer. A total of 150-bp paired end sequencing was performed with a NovaSeq6000 machine (Illumina) at a library concentration of 1.1 nM, generating > 30 M read pairs per sample. All raw sequencing data and associated genotype/phenotype/experimental information is stored in the European Genome-phenome Archive (EGA) under controlled access with Dataset ID EGAS00001005830 [28 ].

Milk samples (n = 3) were identified as being of interest for additional analysis, based on the results of 16S rRNA gene amplicon sequencing, because of a microbiota highly dominated by a single taxonomic group with the low SCC (< 200,000 cells/mL). These samples were further analyzed via shotgun metagenomics. Sequencing libraries were prepared with Nextera XT DNA Flex Library Preparation Kit (Illumina Inc., USA) and Nextera XT Index Kit (Illumina Inc., USA) according to the manufacturer’s instructions. Paired-end sequencing (2 × 150 bp) was performed on a NovaSeq 6000 machine (Illumina Inc., USA) at Genome Quebec (Montreal, Canada).

Production and sequencing of the libraries were carried out by Novogene (https://en.novogene.com) according to their standard procedures. Briefly, sRNA libraries were directly generated from total RNA using TruSeq Small RNA Library Prep Kit (Illumina). The 3’ and 5’ adaptors were sequentially ligated to the RNA prior to reverse transcription and cDNA generation. cDNAs were enriched by PCR to create the indexed double stranded cDNA library. Size selection was performed using 6% polyacrylamide gel. The quantity of the libraries was determined by quantitative real-time PCR and an equimolar pooling of the libraries was performed. The cDNA libraries were sequenced following a single-batch strategy on a NOVASEQ 6000 machine (Illumina).

Total RNA was isolated from the nine testicular tissues with an RNA Nano 6000 Assay Kit (Agilent Technologies, Santa Clara, CA, USA) following the manufacturer’s protocols. The Agilent 2100 bioanalyzer and NanoPhotometer spectrophotometer were adopted to assess RNA integrity and concentration.
Library preparation. Total RNA was used as input material for the RNA sample preparations. Briefly, mRNA was purified from total RNA using poly T oligo-attached magnetic beads, and cDNA was synthesized using mRNA as a template. Selected cDNA library fragments that were preferentially 370–420 bp in length were purified with the AMPure XP system (Beckman Coulter, Beverly, MA, USA). After PCR amplification, the PCR product was purified with AMPure XP beads, and the library was finally obtained. After construction, the library was initially quantified using a Qubit2.0 fluorometer. qRT-PCR was applied to accurately quantify the effective concentration of the library (higher than 2 nM) to ensure the quality of the library.
Transcriptomic sequencing. After the library was quantified, the different libraries were pooled according to the effective concentrations and the target amounts of data produced and sequenced with the Illumina NovaSeq 6000 machine with 150 bp ends read.

Total RNA was extracted from BMDMs using TRIzol reagent (Takara Biotechnology, Japan) according to the manufacturer’s instructions. Raw RNA sequence data were obtained using an Illumina NovaSeq™ 6000 machine. After acquiring the raw data, the fold change (mean of each RNA in the EV group/mean of each RNA in the control group) and P-values were calculated for each RNA. These P-values were used to calculate the false discovery rate (FDR) for each RNA, which was further used as a filter to identify significant RNAs with a fold change ≥ 2 or ≤ 0.5 and an FDR < 0.05. The R 3.5.3 program was used to create the volcanic plots. The 20 most enriched pathways related to signaling transduction are presented and were used to reveal the associated pathways after a pathway analysis with the Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway database. For miRNA analysis in myotube-derived EVs, miRNA was extracted from isolated EVs and used to characterize miRNA profile in skeletal myotube-derived EVs using the above-mentioned method.