GSM4294467: Dvir_zebrafish_Dark_3_010919; Danio rerio; RNA-Seq
Fish were euthanized in MS222 and the stripe and interstiped regions were micro-dissected from fish expressing both pnp4a:palmmCherry and pnp4a:nlsEos. Stripe and interstipe regions were enzymatically dissociated separately with Liberase (0.25 mg/ml in dPBS) at 25°C for 15 min followed by manual trituration with increasingly narrower flame polished glass pipette for 3 min at a time for three times. Cells suspensions were then filtered through a 70 μm Nylon cell strainer to obtain a single cell suspension. Liberated cells were re-suspended in 1% BSA / 5% FBS in dPBS before FACS purification. The flow cytometry experiments were performed on a BD FACSAria II SORP sorter (BD Biosciences, San Jose, CA, USA) coupled with BD FACSDiva software (BD Biosciences) for instrument operation, data acquisition, and analysis. A 637 nm and a 488 nm laser were utilized for fluorophore-excitation and a 100-μm nozzle was used to generate single droplets under 20PSI sheath pressure. The applied settings were as follows: forward light scatter (FSC) detector photomultiplier tube (PMT) gain setting = 80 V with a 1.5 neutral density filter; side light scatter (SSC) detector PMT gain setting = 90 V; FSC threshold = 10,000; PE-Texas Red (PE TX Red) channel PMT gain setting = 280 V; fluorescein isothiocyanate (FITC) channel PMT gain setting = 280 V. Sample dilution and flow rate were adjusted to optimal event recordings for 96 well plate single cell sorts (below 500 processed events per sec). The population of zebrafish skin iridophores was designated based on their FSC and SSC characteristics and back-gating on fluorescence. Control zebrafish skin samples were used to gate out the highly auto fluorescent cells among the members of this population. Fluorescently labeled (PNP4-mCherry-Eos) from either the stripe or interstripe skin samples were sorted separately.  Single cells with high mCherry and Eos expression were collected into 3uL of smart-scrb lysis buffer (0.2% Triton X-100 [Sigma], 0.1 U/ul RNAse inhibitor [NEB]) using „single cell” indexed sorting mode. Five indexed 96 well plates were collected for each skin band. After sorting, the samples were spun down at 3166 rcf at 4ºC for 2 minutes in an Eppendorf tabletop 5810R centrifuge (Eppendorf AG, Hamburg, Germany) and stored at -80ºC until further processing. Four plates of each skin band were selected for sequencing.  cDNA was prepared from sorted cells as described previously (Cembrowski et al., 2018) with minor modifications to the sequencing configuration.  Reverse transcription, PCR, purification, tagmentation, and library quantification were performed as described.
GEO Accession
GSM4294467