title: Shotgun sequencing of hankyphage capsids
design: Shotgun sequencing of hankyphage capsids
library_construction_protocol: Lysates were mixed with PEG8000 1M NaCL 10% (final concentration) to a final volume of 250mL and let precipitate overnight at 4°C. The lysates were then centrifuged at 20,000g for 2-3h, and the pellet was resuspended by pipetting in 1mL of DNase buffer and then placed in a shaker at 37°C for 30 minutes to fully resuspend the pellet. Then 96ul high titre purified phage were mixed with 2ul of turbo DNAase and 2ul of benzonase and incubated at 37°C for 1h. The DNase was then inactivated by adding EDTA 15 mM (final concentration) and a heat-treatment for 10 minutes at 75°C. Then, a proteinase K treatment (3,7 mg/mL final concentration) enhanced with SDS (0.74% final concentration) at 55°C for 45 minutes was performed to degrade phage capsids.  For extraction of the remaining DNA, the PureGene DNA extraction kit (Qiagen) with the protocol for gram negative bacterial DNA extraction (page 60) starting from step 8 (adding directly 100µl of protein precipitation solution to my 100µl sample) was used. An overnight DNA precipitation step was performed at 4°C and the ethanol washes were performed on the next day. DNA was then resuspended for 1 hour at 60°C. For the jmh43::dcas9-repcHP capsids sample, the Novogene NGS DNA Library Prep Set (Cat No.PT004) was used for library preparation. Index codes were added to each sample. Briefly, the genomic DNA was randomly fragmented to size of 350bp. DNA fragments were end polished, A-tailed, ligated with adapters, size selected and further amplified by rolling circle amplification. PCR products were then purified (AMPure XP system), their size distribution was assessed by Agilent 2100 Bioanalyzer (Agilent Technologies, CA, USA), and they were quantified using real-time PCR.