title: FOLLOWUP_P3_H11
organism: Danio rerio
description: Protocols: A single kidney from heterozygote Tg(cd41:EGFP) or wild-type fish was dissected and carefully passed through a strainer using the plunger of a 1-mL syringe. In the follow up experiment, circulating GFP positive cells were collected from the dissected heart of the same fish. Cells were collected in cold 1xPBS/5% FBS. The kidney of a non-transgenic line was used to set up the gating and exclude autofluorescent cells. Dead cells were excluded based on propidium iodide (PI) staining. Individual cells were sorted, using a Becton Dickinson Influx sorter with 488- and 561 nm lasers 26, and collected in a single well of a 96 well plate containing 2.3 uL of 0.2 % Triton X-100 supplemented with 1 U/uL SUPERase In RNAse inhibitor (Ambion). At the same time, information about cell size and granularity, as well as the level of the fluorescence, were recorded. Whole transcriptome amplification and library preparation was performed using the Smart-seq2 protocol 27, 28, with ERCC spike-in controls added at the same time as the oligo-dT and dNTP mixture.