Precooled methanol

Both the sample bacterial preparation and GC-MS analysis were performed as described previously (23 (link)). Briefly, 10 high- and 10 low-virulent strains were cultured in the LB medium with 200 rpm at 37℃ until reaching an optical density at 600 nm (OD600) of 1.0. The aliquot of 10-mL cells was quenched with precooled methanol (Sigma Aldrich) and by ultrasonication. Ribitol (0.1 mg/mL, Sigma Aldrich) was added as an internal standard. The aliquot of the 500-µL supernatant was separated with 12,000 g at 4°C for 10 min and dried by a vacuum centrifugation dryer (Labconco). Methoximation–pyridine hydrochloride (Sigma Aldrich) was added to the dried fraction above and continuously shaken with 200 rpm at 30°C for 90 min. Eighty microliters of N-methyl-N-trimethylsilyltrifluoroacetamide (Sigma Aldrich) was added and incubated at 37°C for 30 min. The data were analyzed using an Agilent 7890A GC and an Agilent 5975C VL MSD detector (Agilent Technologies). The compounds were identified by Agilent Chrom Station software (Agilent Technologies) and the National Institute of Standards and Technology (NIST) library. Every sample was analyzed in duplicate.

For testing the localization of NEK1 and C21ORF2 in cycling cells (Fig 5A), cells were seeded on coverslips (Standard #1.5; Thermo Fisher Scientific) in a 24-well plate at a density of 4 × 10⁴ cells/well for ARPE-19 WT or 6 × 10⁴ cells/well for ARPE-19 NEK1 or C21ORF2–KO cell lines. Cells were grown for 24 h before fixation. For cilia localization, ARPE-19 WT cells (6 × 10⁴ cells/well) were seeded to an eight-well Nunc Lab-Tek Chambered Coverglass (#155361; Thermo Fisher Scientific) for 24 h and serum starved with FBS-free culture medium for 48 h before fixation. For fixation, cells were washed with PBS, fixed for 3 min at room temperature in 3% PFA (Acros Organics) in PBS followed by 5 min permeabilisation at −20°C with pre-cooled methanol (Sigma-Aldrich). Cells were washed three times with PBS before staining. Fixed cells were blocked with 3% BSA (A9647; Sigma-Aldrich) in 0.1% (vol/vol) Triton X-100 (Sigma-Aldrich)/PBS (hereafter called PBX) for 30 min. Samples were incubated with primary antibodies diluted in 3% BSA in PBX for 1 h at room temperature in a humid chamber. After washing three times with PBX, samples were incubated with secondary antibodies in a humid chamber for 30 min. Cells were washed three times with PBS and mounted with Mowiol (Calbiochem).