WGS_FACS_BYxRM_segregants
We isolated genomic DNA from thawed glycerol stocks of the sorted segregant pools for whole-genome sequencing. We centrifuged 800 L of each pool at 3700 rpm for 10 minutes to pellet the cells and discarded the supernatant. To digest cell walls we resuspended the cells in 800 L of 1 M sorbitol, 0.1 M EDTA, 14.3 mM -mercaptoethanol, and 500 U of Zymolyase lytic enzyme (United States Biological) and incubated for 2 hours at 37C on a MixMate at 1100 rpm. We re-pelleted the cells, removed the supernatant, and extracted DNA from the cells using the Quick-DNA 96 Plus kit (Zymo Research), according to the manufacturers instructions, including an overnight protein digestion in 20 mg / mL of proteinase K solution. We eluted the DNA using 35 L of DNA Elution Buffer (10 mM Tris-HCl [pH 8.5], 0.1 mM EDTA) and determined DNA concentration on a Synergy H1 plate reader (BioTek Instruments) in 96-well plates using the Qubit dsDNA BR assay kit (Thermo Fisher Scientific, Waltham, MA, USA).  We prepared the genomic DNA for short-read whole-genome sequencing on the Illumina NovaSeq platform using a previously established approach (Albert et al., 2014; Brion et al., 2020; M. A. Collins et al., 2022, 2023). We used the Nextera DNA library kit (Illumina) in 96 well plates according to the manufacturers instructions, except that we used a 1:20 dilution of the Tagment DNA enzyme in Tagment DNA buffer. After library generation, we quantified the DNA concentration of each sample using the Qubit dsDNA BR assay kit. For each 96 well plate, 10 L of each sample was pooled and 1 mL of that pool was run in a large well on a 2% agarose gel. We extracted and purified the DNA in the 400 to 600 bp region using the Monarch Gel Extraction Kit (NEB) according to the manufacturers instructions. The DNA from each of the gel extractions was quantified and pooled in equimolar amounts, and submitted for sequencing. The University of Minnesota Genomics Center (UMGC) staff performed quality control assays as described in (M. A. Collins et al., 2022, 2023) on the pooled library before sequencing. Briefly, library concentration was determined using PicoGreen dsDNA quantification reagent (Thermo Fisher Scientific), library size was determined using the Tapestation electrophoresis system (Agilent Technologies, Santa Clara, CA, USA), and library functionality was determined using the KAPA DNA Library Quantification kit (Roche, Penzberg, Germany). The submitted pooled library passed each quality control assay. The pooled library was sequenced on the Illumina NovaSeq with 150 bp paired end reads. Of the samples used for analysis, the median number of reads produced per sample was 1,692,391, with the minimum being 304,256 reads and the maximum being 5,874,865 reads. UMGC performed sequence data de-multiplexing.