Genomic sequencing of Histoplasma strain deltaURA5_WU15-Agr-YB T-DNA insertion in WU15 background
Cells from 10 mL of dense liquid culture were collected by centrifugation and kept at 80C until DNA extraction. The cell pellets were thawed and washed in TE buffer, and then resuspended in lysis buffer (50 mM Tris [pH 7.2], 50 mM EDTA, 0.1 M SDS, 0.14 M -mercaptoethanol). Cells were lysed in buffer by bead-beating with zirconia beads, and the samples were incubated at 65C for 1 hour. 800 l of 1:1 phenol-chloroform solution was added to each sample followed by thorough mixing. The samples were centrifuged for 15 minutes at 13,000 rpm. The aqueous fraction was added to 470 l of 0.13 M sodium acetate in 95 % isopropanol and mixed by inversion to precipitate the DNA. The supernatant was discarded after a 2-minute centrifugation, and the pellet was washed with 70 % ethanol and air-dried at 65C. 1 mL TE buffer with 1.5 l RNase A (10mg/ml) was added to each sample and the pellet was dissolved at 65C.  Illumina library preparation and sequencing was performed using a Nextera DNA Flex Library Prep kit followed by sequencing on an Illumina NovaSeq at the UCSF Center for Advanced Technology.