title: Full genome sequencing of H. mephisto isolate P3.3 progeny population
design: For DNA extraction, the Zymo Quick DNA/RNA extraction kit (Catalog number D7003) protocol previously described (Weinstein et al., 2019) was followed. The frozen pellet of 300uL was thawed and added directly to a Zymo ZR BashingBead tube (Zymo catalog number S6012). The tube was taped directly onto the head of a vortexer and run at top speed for 3 min, followed by a 1 min break, and 3 more min vortex. The samples were digested in proteinase K buffer as described in the manufacturer's instructions, with two modifications: the digestion was performed at 37 C, and 20uL of RNAse A (10mg/ml) was added to remove genomic RNA. Zymo Quick DNA/RNA kit has a dual-column flow for isolating both nucleic acids; for this experiment however the RNA extraction was omitted and only DNA was collected. The DNA was eluted from the kit's spin-column into 50uL of nuclease-free water.Once the DNA was extracted it was again RNAse A treated. For this we added 10uL of RNAse A stock (10mg/ml) to the DNA (making 60uL total) and incubated at 37 C for 2 hours. To clean up the DNA we used the Zymo RNA Clean and Concentrator-5 (catalog number R1015) and the manufacturer's instructions were followed for isolating large nucleic acid fragments. Briefly, the RNA binding buffer and 100 percent ethanol were pre-mixed prior to addition of sample and loading onto the column. The samples were processed following manufacturer's instruction including two washes with RNA wash buffer (ethanol) and a dry spin of 2 min prior to elution to remove residual wash buffer. Final DNA was eluted in 50uL of nuclease-free water.The genomic DNA, prepared as described above was converted into Sequencing libraries using the Rapid Barcoding Kit (SQK-RBK114.24) following the manufacturer's protocol. Sequencing used multiple Flongle flowcells to obtain sufficient coverage; the data from these flowcells has been merged into one fastq file.
library_name: P3.3-library1