Observer z1 inverted fluorescent microscope

Dummy and ERCC2 KO cells were generated from K562 cells with the sgRNA sequences listed in Table S6. As a positive control, K562 cells were exposed to UV light for 1hr in a cell culture hood. Male WT mice (n=3 each condition) were exposed to 21% or 80% O₂ for 5 days. Mice were sacrificed following isoflurane anesthesia. Single cells from the lung were isolated from the lungs as described previously ^{111 (link)}. The cell pellet was resuspended in ice cold PBS for the comet assay.
Cellular DNA damage was measured by the comet assay in alkaline solution (Abcam 238544) according to the manufacturer’s instructions. The slides for in vitro and in vivo samples were imaged with a Nikon Spinning Disk epifluorescent microscope and a Zeiss Observer Z1 inverted fluorescent microscope, respectively. At least 35 randomly selected nucleoids were analyzed per in vitro sample and at least 150 nucleoids were analyzed per mouse sample using Comet Score 2.0 software. Data are presented as percent of DNA in the comet tail; the intensity of the comet tail relative to the comet nucleoid head indicates the extent of DNA damage, including DNA single- and double-strand breaks, alkali labile DNA adducts, and the majority of apurinic/apyrimidinic sites. The experiment was performed in 3 independent biological replicates.