title: Illumina MiSeq sequencing
design: TnSeq of rugose colonies of Vibrio cholerae co969 grown at 22ºC, for the identification of potential biofilm repressors at low temperature.
library_construction_protocol: Transposon mutagenesis was performed by conjugation of V. cholerae co969 (recipient strain) with E. coli SM10 λ pir (pSC189) (Chiang & Rubin, 2002) (donor strain), as described by Dörr et al. (Dorr et al, 2016). Transposon mutants were selected on LB plates (10 g/l NaCl) containing both streptomycin (200 µg/ml) and kanamycin (50 µg/ml). For the screen for biofilm repressors at 22ºC, 459 out of 11000 screened mutants were selected due to their rugose colony morphology at 22ºC. To identify the genes interrupted by the transposon, the selected mutants were pooled together and transposon insertion sequencing (Tn-seq) was performed as described previously (Chao et al 2013). In brief, the chromosomal DNA of the pooled mutants was purified and sheared by sonication (Covaris sonicator E220). The overhanging ends were blunted using a blunting enzyme kit (NEB) and an A-tail was added with Taq DNA polymerase (NEB). Then, Illumina adaptors were ligated (with T4 DNA ligase, NEB) and the genomic DNA-transposon junctions were amplified (with Phusion® High-Fidelity DNA polymerase, NEB). Sequencing was performed using an Illumina MiSeq benchtop sequencer (Illumina, San Diego, USA). Data analysis for determination of the TA insertion sites was conducted as described previously (Chao et al, 2013; Pritchard et al, 2014). For visualization of transposon insertion profiles, Sanger Artemis Genome Browser and Annotation tool was used (Rutherford et al, 2000).