Truseq nano dna library prep

Samples were induced as described in previous sections. At the indicated time points after MC-induction, 12 ml of sample was collected and DNA extraction was performed using the ‘ChargeSwitch® gDNA Mini Bacteria Kit’ from Thermo Fisher Scientific, following the manufacturer’s instructions. The DNA was precipitated by 0.3 M NaOAc and 2.25 volume of 100% ethanol, then pelleted at 12,000 × g for 30 min at 4 °C and washed once with 1 ml of 70% ethanol. After centrifugation, the DNA pellets were air dried for 30 min and resuspended in 50 μl nuclease free water. Quality control of DNA samples was tested using Agilent Bioanalyzer 2100 and whole genome sequencing (WGS) was performed at the University of Glasgow Polyomics Facility using Illumina TruSeq DNA Nano library prep, obtaining 2 × 75 bp pair end reads with DNA PCR free libraries. A total of 2.2 M reads were generated and trimmed reads were mapped to the appropriate genome: P22 (NC_002371.2), ES18 (NC_006949) and LT2 (NC_003197). Only one replicate was sequenced per experiment.

Overnight cultures were prepared by inoculating a single colony from each strain into a 5 ml TSB and incubated at 37 °C, 120 rpm for 16–18 h. The desired strains were then diluted 1:50 in 13 ml of fresh TSB and grown to exponential phase (OD₅₄₀~0.15–0.20) to collect samples before induction. Next, the cultures were treated with MitC (2 μg ml⁻¹) and incubated at 30 °C and 80 rpm for 60 min prior to sample collection. One ml samples were collected, and genomic DNA was extracted using the GenElute Bacterial DNA kit (Sigma Aldrich) according to the manufacturer’s instructions. The DNA was precipitated by adding 10% (v/v) 3 M sodium acetate (pH 5.2), 2.5 volumes of 100% ethanol and incubation of the mixture for 1 h at −80 °C. The DNA was then pelleted at 16873 × g for 30 min at 4 °C and washed once with 1 ml of ice-cold 70% (v/v) ethanol. After centrifugation, the DNA pellets were air-dried for 30 min and re-suspended in 25 μl of TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 8.0). Quality control of DNA samples was tested using Agilent Bioanalyzer 2100 and whole genome sequencing (WGS) was performed at the University of Glasgow’s Polyomics Facility using Illumina TruSeq DNA Nano library prep, obtaining 2 × 75 bp pair end reads with DNA PCR free libraries.

RNA for transcriptome sequencing was extracted from the 20 pooled microdissected HF of the same cycle stage and the IFE collected from each dog. RNA extraction, library preparation and RNA-seq were performed as described in a previous study [17 (link),36 (link)]. In brief, total RNA was extracted using the RNeasy Micro Kit (Qiagen, Hombrechtikon, Switzerland) including proteinase K digestion according to the manufacturer’s protocol. Total RNA content was measured using the Qubit^® 2.0 Fluorometer (Invitrogen, Thermo Fisher, Basel, Switzerland) and RNA quality was assessed with a Bioanalyzer (Agilent 2100; Agilent Technologies, Basel, Switzerland). High quality RNA (RIN > 9) was reverse transcribed into cDNA with the SMART-Seq v4 Ultra Low Input RNA Kit (Takara, Saint-Germain-en-Laye, France) and libraries were constructed following the TruSeq^® Nano DNA Library Prep (Illumina, San Diego, CA, USA) protocol for RNA sequencing. Multiplexed total cDNA libraries were sequenced on two lanes using an Illumina HiSq3000 instrument with 100 bp single-end sequencing cycles. On average, thirty million reads were collected, converted into FASTQ file format and demultiplexed.
The data are available from the European Nucleotide Archive (ENA), study accession number PRJEB21761 and sample accessions SAMEA7016531-SAMEA7016551 [50 ].