Wallac 1420 workstation

The identified BAC clones were picked to streak in fresh LB medium plates (Fisher Scientific) with 12.5 μg/ml chloramphenicol and the plates were cultured at 37°C overnight. A single colony from the overnight culture was inoculated into 2 ml fresh LB medium containing 10 g tryptone, 5 g yeast extract, and 10 g NaCl (Fisher Scientific) with 12.5 μg/ml chloramphenicol and grown with vigorous shaking (300 rpm) to late logarithmic phase (∼8 h) at 37°C. Then, 16 μl of the starter culture was added to 8 ml LB liquid medium in a larger volume vessel and grown with vigorous shaking at 300 rpm at 37°C overnight (16–18 h).
BAC DNA was extracted from the overnight liquid culture following the method of R.E.A.L. Prep 96 Plasmid kit (Qiagen). DNA yield was determined by Quant-iT^TM dsDNA Assay Kit (Invitrogen) with fluorescein (485 nm/535 nm) machine Wallac 1420 workstation (PerkinElmer, Inc., Turku, Finland). DNA quality was detected by using HindIII digestion of the BAC DNA at 37°C for over 2 h and checked on 1% agarose gel. Approximately 1–2.5 μg DNA was required for each clone sequencing.

CellTiter-Glo Luminescent (CTG) Cell Viability Assay (Promega, Madison, WI, USA) was used to assess the relative rate of cell proliferation by measurement of ATP content present in cells according to instructions provided by the manufacturer. Briefly, cells were washed three times in HBSS and resuspended in experiment media as previously described. A total of 1 × 10⁴ cells were seeded in 100 μL medium per well in a 96-well optical plate. Experiments were run at 37°C in 5% CO₂. After 24 h, the provided assay reagent was added to the wells, after which the plate was agitated on a microplate shaker for 2 minutes. The plates were subsequently kept at room temperature for 10 minutes before luminescence was quantified. The luminescent signal was recorded with a Victor3 plate reader and Wallac 1420 Workstation software (PerkinElmer Inc.).