XB-ART-57511Dev Dyn September 1, 2021; 250 (9): 1381-1392.
Tissue disaggregation and isolation of specific cell types from transgenic Xenopus appendages for transcriptional analysis by FACS.
BACKGROUND: 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. RESULTS: 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. CONCLUSIONS: Here we gate against both nontransgenic 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.
PubMed ID: 33137227
PMC ID: PMC8088453
Article link: Dev Dyn
Genes referenced: actc1 atoh1 bhlha15 camk4 carm1 ccdc40 dnajb9 eef1a1 eef2k eif4e fem1b fgf2 gap43 gfi1b hoxb1 iqcb1 mks1 nos1 pax6 pls3 psmb2 ptprd rab21 sim1 slc7a6os smarcc2 spns2 sypl2 tshz3 ube2j1 vapa zeb2 zfr
GO keywords: regeneration
GEO Series: GSE158331: NCBI
References [+] :
Aztekin, Identification of a regeneration-organizing cell in the <i>Xenopus</i> tail. 2019, Pubmed, Xenbase