Tissues used for real-time RT-PCR assay were soaked in RNAlater solution overnight in 4°C before removing the solution and freezing the samples in −80°C.
To ensure a homogenous sampling of the instillate, a volume of 2 ml was aspirated and reintroduced into the airspaces three times before removing each sample.
These include failure to pick the worst-case houses, not allowing water to stand long enough before sampling, removing the aerator (a screen added to the faucet to reduce spray and/or conserve water) before sampling, and sampling in cooler months (when lead concentrations in water are lower because lead dissolves less readily in chilled water).
Using PLINK (Purcell et al. 2007), we removed SNPs with >20% missing data before removing samples with >20% missing data.
After reaction at 30 °C with agitation at 200 rpm for 20 min, 100 μL of 1 M HCl was added to stop the reaction, and centrifugation was performed at 13,000×g for 10 min, before removing 500 μL of sample for reverse-phase high-performance liquid chromatography analysis using a Zorbax® SB-Aq column (250 × 4.6 mm, 5 µm; Agilent Technologies, USA) at a detection wavelength of 210 nm (Ni et al. 2013).
Excess sample was then removed before the samples on the stubs were sputter-coated with gold palladium, and finally examined with a Leica S440 Scanning Electron Microscope (for DMSO-induced pigments) [27] and a Hitachi S-4200 (Japan) Scanning Electron Microscope (for PEG-induced pigments) [30].
The sample was evaporated to dryness to ensure that all methanol was removed from the sample before it was redissolved in 0.5 mL n-hexane.
Potential telephone numbers were drawn from commercially available Electronic White Pages, all duplicate, cellular and business numbers were removed from the sample before each survey commenced.
This latter step ensured that all cumulus cells were removed from the sample before extraction of genomic DNA, which involved a 5 hour treatment with Proteinase K followed by ethanol precipitation.
