ATAC-seq analysis to examine global accessibilty of chromatin in limb bud cells

The study compares two cell populations one where Etv2 is expressed and other lacks Etv2 expression. The cells from wild-type E9.5 hindlimbs , cells from Shh-EGFP E10.5 anterior hindlimbs , EGFP+ cells from Shh-EGFP E10.5 posterior hindlimbs, and EYFP+ cells from Etv2-EYFP transgenic E10.5 hindlimbs and forelimbs were collected and processed Overall design: Limb bud tissues were digested in 200 ml of TripLE (ThermoFisher 12605010) at 37°C in two cycles of 5 min incubation followed by trituration. Cells were cooled on ice for 5 min between incubations. Digestion was stopped with 1ml of medium with 10% fetal calf serum that was filtered through a 50 mm mesh filter (Partec). Cells were collected by centrifugation and stained with staining medium containing 0.2 mg each of CD31-APC, Tie2-APC, CD45-APC and 0.5 mg anti CD16/CD32, and EYFP positive, APC negative live cells were sorted on FACSAria (BD). Cells from EYFP negative embryos were used as a negative control for FACS sorting.