run_metadata: 56588

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rowid	run.accession	experiment.accession	sample.accession	study.accession	bioproject	study.title	study.alias	study.type	study.abstract	study.attributes	study.PMIDs	sample.description	sample.title	sample.alias	sample.centername	sample.attributes	GEOsample.title	GEOsample.dataprocessing	GEOsample.source	GEOsample.treatmentprotocol	GEOsample.extractprotocol	GEOsample.growthprotocol	GEOsample.characteristics	GEOsample.accession	experiment.title	experiment.alias	experiment.library_name	experiment.design_description	experiment.library_construction_protocol	experiment.attributes	experiment.library_strategy	experiment.library_source	experiment.library_selection	experiment.library_layout	experiment.platform	experiment.instrument_model	experiment.spot_descriptor	experiment.study_ref	run.title	run.attributes	run.filename	run.semantic_name	run.total_bases	run.total_spots	run.alias	run.read_lengths	run.base_counts	run.r1_length	run.r2_length	run.r3_length	run.r4_length	run.Acount	run.Ccount	run.Gcount	run.Tcount	run.Ncount	run.experiment	run.pool_member	submission.accession	submission.srasource	submission.bioprojectsource	seqdetective.n_mates	seqdetective.mapping_rate.mate1	seqdetective.mapping_rate.mate2	seqdetective.nofeature_rate.mate1	seqdetective.nofeature_rate.mate2	seqdetective.sparsity.mate1	seqdetective.sparsity.mate2	seqdetective.pos_strand_rate.mate1	seqdetective.pos_strand_rate.mate2	seqdetective.readlen.mate1	seqdetective.readlen.mate2	seqdetective.judgement.mate1	seqdetective.judgement.mate2	seqdetective.judgement.reason	platform_family	instrument_generation	read_bias	selection_class	prep_kit	sc_or_bulk	tech_class	technology	tech_variant	submission.bioprojectsource.country	earliest_date	devstage_curation	devstage_curation_coarse	tissue_curation	tissue_curation_coarse
56588	SRR11015122	SRX7670821	SRS6098554	SRP247126	PRJNA604753	IN SITU DIFFERENTIATION OF IRIDOPHORE CRYSTALLOTYPES UNDERLIES ZEBRAFISH STRIPING	GSE144734	Transcriptome Analysis	Skin color patterns are ubiquitous in nature evolve rapidly and impact social behavior predator avoidance and protection from ultraviolet irradiation. A leading model system for vertebrate skin patterning is the zebrafish; its alternating blue stripes and yellow interstripes depend on guanine crystal containing cells called iridophores that reflect light. It was suggested that the zebrafish's alternating color pattern arises from a single type of iridophore migrating differentially to stripes and interstripes. When we tracked iridophores however we found they did not migrate between stripes and interstripes but instead differentiated and proliferated in place based on their micro environment. RNA sequencing analysis further revealed that stripe and interstripe iridophores had different transcriptomic states with many differences in gene expression and pathway enrichment while cryogenic scanning electron microscopy and micro X ray diffraction identified different guanine crystal organizations and responsiveness to norepinephrine all indicating that stripe and interstripe iridophores are different cell types. Based on these results we present a new model of skin patterning in zebrafish in which distinct iridophore crystallotypes containing specialized physiologically responsive subcellular organelles arise in stripe and interstripe zones by in situ differentiation. In this model pattern phenotype depends not only on interactions among pigment cells that affect their arrangements but also on factors that specify subcellular organization and physiological responsiveness of specialized organelles. Overall design: Unbiased classification of iridophores based solely on their RNA profiles obtained by single cell RNA sequencing		pubmed:33319779		Dvir zebrafish Light 6 010919	GSM4294470		source name:Flank dermis\|tissue:Flank dermis\|stripe pattern:light\|reporter:Adult pnp4a:palmmCherry and pnp4a:nlsEos postive cells	Dvir zebrafish Light 6 010919	The libraries were pooled and sequenced on an Illumina NextSeq 550 high output flowcell with 26 base pair read1 50 base pair read 2 8 base pair i7 index. Samples were demultiplexed using bcl2fastq v2.20 software from Illumina with default settings. Barcode and UMI sequences were extracted from read 1 using custom python scripts. Barcode error correction was done using starcode v1.1 Zorita et al. 2015 with the following additional parameters: “ d 1 q print clusters”. Read 2 sequences were renamed using the error corrected barcode from starcode and UMI sequences from read 1 and were aligned to the Danio rerio GRCz11.94 genome assembly from Ensembl ensemble.org using STAR Dobin et al. 2013 with the following additional parameters: “ alignIntronMax 200000 outSAMattributes All outSAMunmapped Within outSAMtype BAM Unsorted”. An alignment is considered valid if it aligns to an exon feature on the correct strand and is unique. Valid alignments that had at least 50% of the read aligned to an exon feature were used to create gene level counts using a custom python script to collapse UMIs by gene. All custom scripts are available by request. Genome build: Danio rerio GRCz11.94 genome assembly from Ensembl.org Supplementary files format and content: single Microsoft Excel file that includes counts per million CPM and correct p values for each sample and gene	Flank dermis	Untreated fish	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. post 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.	Zebrafish were housed at 28 °C 14L:10D fed rotifers Artemia and flake food	tissue:Flank dermis\|stripe pattern:light\|reporter:Adult pnp4a:palmmCherry and pnp4a:nlsEos postive cells	GSM4294470	GSM4294470: Dvir zebrafish Light 6 010919; Danio rerio; RNA Seq	GSM4294470		1	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. post 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:GSM4294470	RNA-Seq	TRANSCRIPTOMIC	cDNA	PAIRED	ILLUMINA	NextSeq 550		SRP247126			Plate_298_Q7_1.fastq.gz Plate_298_Q7_2.fastq.gz	fastq fastq	2559768084.0	33681159.0	GSM4294470 r1	0:26 1:50	A:716945728;C:505642244;G:535898808;T:800467367;N:813937	26	50			716945728	505642244	535898808	800467367	813937	SRX7670821	SRS6098554	SRA1037187	GEO	JLS, HHMI Janelia Research Campus	2	0.00886	0.70125	0.00828	0.44288	0.99829	0.85965	0.50943	0.5945	26	50	T	B	sc-like readlen	illumina	nextseq	unknown	cdna_unspecified	unknown	sc_generic	single_cell_generic	generic-scrnaseq-only		United States	2020-02-04	Adult	Adult	Skin	Surface Structure