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 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.

本研究对比了两类细胞群：一组表达Etv2，另一组不表达Etv2。研究者收集并处理了以下样本：野生型E9.5期小鼠后肢细胞、Shh-EGFP转基因小鼠E10.5期后肢前区细胞、Shh-EGFP转基因小鼠E10.5期后肢后区的EGFP阳性细胞，以及Etv2-EYFP转基因小鼠E10.5期后肢与前肢的EYFP阳性细胞。将肢芽组织置于200 mL TripLE（赛默飞世尔，货号12605010）中，于37℃下分两轮消化：每轮孵育5分钟后进行吹打分散，两次孵育之间将细胞置于冰上冷却5分钟。使用1 mL含10%胎牛血清的培养基终止消化，随后通过50 mm孔径滤网（Partec公司）过滤。离心收集细胞后，使用含CD31-APC、Tie2-APC、CD45-APC各0.2 mg，以及抗CD16/CD32抗体0.5 mg的染色液进行染色。通过FACSAria流式细胞分选仪（BD公司）分选出EYFP阳性且APC阴性的活细胞，以EYFP阴性的胚胎来源细胞作为荧光激活细胞分选（FACS）的阴性对照。