run_metadata: 25109

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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
25109	SRR25822232	SRX21332628	SRS18578260	SRP454539	PRJNA1004255	Unique activities of two overlapping PAX6 retinal enhancers	GSE240575	Transcriptome Analysis	Enhancers play a critical role in development by precisely modulating spatial temporal and cell type specific gene expression. Sequence variants in enhancers have been implicated in disease however establishing the functional consequences of these variants is challenging due to a lack of understanding of precise cell types and developmental stages where the enhancers are normally active. PAX6 is the master regulator of eye development with a regulatory landscape containing multiple enhancers driving expression in the eye. Whether these enhancers perform additive redundant or distinct functions is unknown. Here we describe the precise cell types and regulatory activity of two PAX6 retinal enhancers HS5 and NRE. Using a unique combination of live imaging and single cell RNA sequencing in dual enhancer reporter zebrafish embryos we uncover differences in the spatiotemporal activity of these enhancers. Our results show that although overlapping these enhancers have distinct activities in different cell types and therefore likely non redundant functions. This work demonstrates that unique cell type specific activities can be uncovered for apparently similar enhancers when investigated at high resolution in vivo. Overall design: In order to define the precise cell types within the retina where the PAX6 enhancers HS5 and NRE are active we carried out scRNA seq on eyes from NRE eGFP/HS5 mCherry zebrafish reporter embryos. With this technique we aimed to uncover cell type or transcriptional differences between the two enhancer active populations. We dissected eyes from 48 hpf NRE eGFP/HS5 mCherry embryos and used FACS to enrich for either mCherry positive/HS5 active cells or eGFP positive/NRE active cells. Three samples for each population were processed for scRNA seq using the 10x Genomics Chromium single cell three prime gene expression technology.		pubmed:37643867		gfp enriched rep3	GSM7702835		source name:Eye\|tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf\|geo loc name:missing\|collection date:missing	gfp enriched rep3	Cell Ranger v6.1.2 was used to perform alignment filtering barcode counting and UMI counting. A custom reference genome was created for alignment using cellranger mkref combining the Danio rerio GRCz11 genome assembly with manually annotated eGFP and mCherry sequences. Cell calling and QC: The emptyDrops function from DropletUtils was used to filter out empty droplets/barcodes not corresponding to cells Lun et al. 2019. Mitochondrial and ribosomal genes were excluded from the emptyDrops analysis to improve the filtering of droplets containing ambient RNA or cell fragments. The scater package was used to filter cells based on the QC metrics of library size detected genes and mitochondrial reads McCarthy et al. 2017. Cells with detected genes ≥ 500 library size ≥ 800 and mitochondrial reads ≤ 10% were retained. Within the processed dataset mean reads per cell = 11239 and median genes per cell = 1554. Reference mapping and filtering: The SingleR package was used to annotate cell types based on mapping to the zebrafish single cell transcriptome atlas Aran et al. 2019; Farnsworth et al. 2019. Expression matrix and cell annotation data were downloaded from the UCSC cell browser http://zebrafish dev.cells.ucsc.edu; only the 2 dpf data were used for mapping. Erroneously sorted cells of non retinal identity for example pigmented cell types such as melanocytes with high autofluorescence were filtered out at this stage. This was carried out to improve the resolution of clustering for retinal cell types. Clustering and cell type annotation: Seurat v4 was used for clustering and further analysis for a total of 6 288 cells Butler et al. 2018. SCTransform was used to perform log normalisation scaling and highly variable gene HVG detection on a dataset consisting of the 6 samples merged into one. Standard SCTransform options were used with regression of mitochondrial expression and cell cycle stage using ‘vars.to.regress’. We performed Principle Component Analysis PCA on the normalized counts matrix restricted to HVGs using Seurat's RunPCA function with number of PCs = 50. To enable integration of the samples we then used Harmony to generate PCs corrected for batch effects between libraries Korsunsky et al. 2019. The Harmony PCs were then used to perform K nearest neighbour analysis k=20 and Louvain clustering using Seurat 15 dimensions and resolution 0.6. Clusters were annotated as retinal cell types based on the highest expressed marker genes and other known genes for each cell type using information from the literature and ZFIN Sprague et al. 2008. Cell cycle scoring was performed using the Seurat CellCycleScoring function using zebrafish genes homologous to the ‘s.features’ and ‘g2m.features’ genes provided by Seurat. Assembly: GRCz11 Supplementary files format and content: Tab separated values files matrix files Seurat object RDS file	Eye		NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell 3’ gene expression technology v3.1.		tissue:Eye\|genotype:NRE eGFP/HS5 mCherry\|developmental stage:48 hpf	GSM7702835	GSM7702835: gfp enriched rep3; Danio rerio; RNA Seq	GSM7702835 r1	GSM7702835	1	NRE eGFP/HS5 mCherry embryos were collected and treated with PTU from 12 hpf. At 48 hpf embryos were anaesthetised with Tricaine 20–30 mg/l and placed into Danieau's solution. Eyes were dissected from 100 150 embryos using fine forceps Dumont #5SF and immediately placed into Danieau's solution on ice. Samples were centrifuged at 300g for 1 minute at 4°C then washed with Danieau's solution. Washing step was carried out three times with Danieau's solution and once with FACSmax Amsbio. In a final 500 µl FACSmax the samples were passed through a 35 µm cell strainer to obtain single cell suspension on ice. Samples were sorted for mCherry and eGFP fluorescence using a FACS Aria II BD or CytoFLEX SRT Beckman Coulter machine. Forward and side scatter sorting was used to select single cells from clumps and debris and DAPI staining was used to exclude dead cells. Libraries were prepared using the 10x Genomics Chromium single cell three prime gene expression technology v3.1.		RNA-Seq	TRANSCRIPTOMIC SINGLE CELL	cDNA	SINGLE	ILLUMINA	NextSeq 2000		SRP454539		loader:fastq load.py	2707_GFP_pos_S4_L001_R2_002.fastq.gz 2707_GFP_pos_S4_L001_R1_002.fastq.gz 2707_GFP_pos_S4_L001_I2_002.fastq.gz 2707_GFP_pos_S4_L001_I1_002.fastq.gz	fastq fastq fastq fastq	21424338264.0	155248828.0	GSM7702835 r3	0:10 1:10 2:28 3:90	A:3928077020;C:3077672525;G:3451667186;T:3513734857;N:1242932	10	10	28	90	3928077020	3077672525	3451667186	3513734857	1242932	SRX21332628	SRS18578260	SRA1702612	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	Wendy Bickmore, MRC Human Genetics Unit, University of Edinburgh	1	0.9433		0.14242		0.78328		0.52384		90		B		usable mapping rate	illumina	nextseq_v2	3prime	cdna_unspecified	unknown	sc	single_cell_droplet	10x		United Kingdom	2023-08-10	Hatching	Embryo	Eye	Sensory System