GSE158331
Tissue disaggregation and isolation of specific cell types from transgenic Xenopus appendages for transcriptional analysis by FACS
Xenopus embryos and tadpoles are versatile models for embryological, cell biological, and regenerative studies. Genomic and transcriptomic approaches have been increasingly employed in these frogs. Most of these genome-wide analyses have profiled tissues in bulk, but there are many scenarios where isolation of single cells may be advantageous, including isolation of a preferred cell type, or generation of a single-cell suspension for applications such as scRNA-Seq. Here we present a protocol for the disaggregation of complex tail and limb bud tissue, and use cell type-specific fluorescence in transgenic X. tropicalis appendages to isolate specific cell populations using fluorescence activated cell sorting (FACS). Our protocol addresses a specific challenge in Xenopus embryos and tadpoles: the storage of maternal yolk platelets in each cell, which can introduce light scatter and thereby false positives into FACS analysis. Here we gate against both non-transgenic and ubiquitously transgenic animals to reduce both false positives and false negatives. We use the Xtr.Tg(pax6:GFP;cryga:RFP;actc1:RFP)Papal transgenic line as a test case to demonstrate that nucleic acid preparations made from sorted cells are high quality and specific. We anticipate this method will be adaptable to study various cell types that have transgenic reporter lines to better profile cell types of interest. Overall design: The posterior third of an anestitized tadpole tail was amputated at NF stage 41. Tadpoles were replaced in media without anesthetiz and allowed to regenerated until desired collection timepoint. The regenerated tail was isolated from the tadpole and cells were dissociated into single cell suspension using Liberase, washed in PBS and sorted for fluoresence through an Aria III cell sorter.