Supplementary Material for: Cytogenetic Characterization and Fluorescence in situ Hybridization of (GATA)<sub>10</sub> Repeats on Established Primary Cell Cultures from Indian Water Snake <i>(Natrix piscator)</i> and Indian Mugger <i>(Crocodylus palustris)</i> Embryos

Sex determination among reptiles has continued to draw the attention of geneticists and the mechanisms involved have been extensively studied and documented in the past 3 decades. The setting up of primary cell lines of reptilian tissues is an important tool in the present study which is a unique aspect not applied in earlier studies. Establishing the cell lines from various species of reptiles would help in our understanding of the mechanisms of evolution and differentiation of sex chromosomes. Therefore, in the present study, we have established for the first time primary cell cultures from Indian water snake <i>(Natrix piscator)</i> and Indian mugger <i>(Crocodylus palustris)</i> embryos. In the preliminary growth stage, 2 types of cells, fibroblast- and epithelial-like, were found to be attached and proliferating in vitro. These fibroblast-like cell cultures were later overtaken by epithelial cells. The cell lines were grown in minimal essential medium supplemented with fetal bovine serum and subcultured for a period of 8–10 months. The morphology of cell types was kept under constant observation microscopically. Interestingly, at a subsequent passage of the cells sporadically scattered neuronal-like and beating cells were observed. The suitable temperature for growth of these cell cultures was 28–30°C. Chromosome analysis was performed from the actively proliferating cells, which revealed 5 pairs of macrochromosomes and 15 pairs of microchromosomes in <i>Natrix piscator,</i> and 15 pairs of only macrochromosomes in <i>Crocodylus palustris</i>. (GATA)<sub>n</sub> repeats are well known to be associated with sex chromosomes. Fluorescence in situ hybridization performed with (GATA)<sub>10</sub> repeats delineated the W chromosome in the cells of <i>Natrix piscator</i> which has so far not been reported. This cell culture method has presently only been applied to water snakes and crocodile embryos in the current study, but it will be employed in other reptilian species and could go a long way to being a sustainable source of primary cells. This would eventually serve as an important tool for molecular studies in reptiles and other species in the future.