NextSeq 500 sequencing; SNP analysis of ∆motV/∆motW suppressor of Vibrio cholerae
SNP analysis of ∆motV/∆motW suppressor of Vibrio cholerae
pellet 1 mL of overnight culture in LB GENELUTE Bacterial Genomic DNA Kit (Sigma) 5 µg of genomic DNA in 130 µL H2O was sheared with a Covaris S220 focused ultrasonicator (Covaris, Wobum, MA) with target peak at 500 bp. After blunting the DNA ends with T4 DNA polymerase (New England Biolabs, Ipswich, MA) and Klenow Enzyme (Roche, Basel, Switzerland) followed by A-tailing with Klenow Fragment (New England Biolabs), adaptor DNA (Lioy et al., 2018) was ligated. DNA fragments ranging 400~900 bp in size were purified using Pippin Prep (SAGE Science, Beverly, MA) then amplified with Illumina primers PE1.0 and PE2.0. PCR products were purified using QIAGEN MinElute columns (QIAGEN, Hilden, Germany) then size-selected using AMPure XP beads (Beckman Coulter, Brea, CA). [Lioy VS, Cournac A, Marbouty M, Duigou S, Mozziconacci J, Espéli O, et al. Multiscale Structuring of the E. coli Chromosome by Nucleoid-Associated and Condensin Proteins. Cell. 2018;172: 771-783.e18. doi:10.1016/j.cell.2017.12.027]