Purification of Fluorescent Labeled Cells from Dissociated Ciona Embryos
Genome-wide studies in Ciona often require highly purified cell populations. In this methods chapter, we introduce multi-channel combinatorial fluorescence activated cells sorting (FACS) and magnetic-activated cell sorting (MACS) as two sensitive and efficient tools for isolating lineage-specific cell populations from dissociated Ciona embryos and larvae. We present isolation of trunk ventral cell (TVC) progeny as the test case most commonly used in our laboratory. These approaches may also be applied to purify other cell populations with the proper combination of tissue-specific reporters.
KeywordsTVC Multi-channel combinatorial FACS MACS RNA extraction Cell dissociation Cell-lineage specific population Mosaic expression
We are grateful to Pui-Leng Ip, Ken Birnbaum, and the personnel of GenCore at NYU for their expert assistance with the FACS, ScreenTape, and high-throughput sequencing. We thank Nicole Kaplan for sharing the image of Mesp > tagRFP, MyoD905 > GFP, and Hand-r > tagBFP labelled Ciona late tailbud. Research in the laboratory of L.C. is supported by R01 awards HL108643 and GM096032 from the NIH/NHLBI and NIH/NIGMS respectively; and by grant 15CVD01 from the Leducq Foundation. C.R. has been supported by a long-term fellowship ALTF 1608-2014 from EMBO.
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