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Sample GSM5678000

Query DataSets for GSM5678000

Status

Public on Apr 27, 2023

Title

20126: Zprox1ab_4dpf scRNA-seq

Sample type

SRA

Source name

embryo

Organism

Danio rerio

Characteristics

tissue: embryo
transgenic: Tg(fli1a:negfp);Tg(-5.2lyve1b:dsRed)
facs: nEGFP and dsRed2 double positive
Stage: 4dpf
genotype: Zprox1ab_mutant
platform: 10X Chromium scRNA
version: Single cell 3' V3
facility: Peter Mac Molecular Genomics
cellranger_pipeline: 3.1.0
r_version: 4.0.2
internal_id: 20126
internal_name: Prox1a_b_Mut

Treatment protocol

All injections were performed as previously described [10]. CRISPR genome editing for tspan18a/b was performed as previously described [11], and all genotyping confirmed using PCR [12]. slc7a7aBAC:slc7a7a-Citrineuom10 and fabp11aBAC:fabp11a-Citrineuom10 recombineering was performed as previously described.

Growth protocol

Zebrafish work was conducted in compliance with animal ethics committees at University of Queensland and Peter MacCallum Cancer Centre. The uq52bh (dut) mutant was isolated in a previously described genetic screen [7]. The genetic mapping approach was performed as previously described [8, 9].

Extracted molecule

total RNA

Extraction protocol

To dissociate embryos and obtain single cell suspensions, we followed published protocols [13]. To dissociate embryos and obtain single cell suspension, at the desired developmental stage we deyolked embryos by pipetting up and down and rinsing in Calcium free ringers solution, we centrifuged at 2000rpm for 5’ at 4C and remove supernatant and dissociated the cells by incubating in liberase [2.5mg/mL] (Cat #5401119001 Sigma-Aldrich) diluted at a 1:35 ratio in DPBS at 28.5C for approximately 5’, homogenizing the samples during and after the incubation. To stop the reaction, we added CaCl2 to a final concentration of 1-2mM and FBS to a final concentration of 5-10%. We centrifuged at 2000rpm for 5’ at 4C. discarded the supernatant, in order to be able to asses live vs. dead cells, we re-suspend the cells solution in Zombie Violet TM Viability Dye (Cat# 423113, BioLegend) and incubated for 20’ at RT softly rocking, we rinsed the cells by centrifuging and resuspending in DPBS/EDTA, for ATAC-Seq experiments samples were resuspended in 2%BSA/PBS. Suspension was filtered through a strainer and taken to the FAC sorting facility. In the Flow Cytometry facility, we used the BD FACSAria Fusion sorter (BD Biosciences), we based the selection for the desired population on FSC and SCC, alive cells were selected based on the Zombie Violet profile and double positive cells for the desired transgenics were targeted according to the expression profiles of single cells. Double positive cells were sorted in 300uL 100% FBS in a cold block and taken immediately to the sequencing facility.
Single cell suspensions were sorted by FACS, spun down to concentrate and a cell count was perfomed to determine post-sort viability and cell concentration. Single cell suspension was partitioned and barcoded using the 10X Genomics Chromium Controller (10X Genomics) and the Single Cell 3' Library and Gel Bead Kit (V2; 10X Genomics; PN-120237). The cells were loaded onto the Chromium Single Cell Chip A (10X Genomics; PN-120236) or B (10X Genomics; PN-1000073 or PN-1000074) to target 10,000 cells. GEM generation and barcoding, cDNA amplification, and library construction was performed according to the 10X Genomics Chromium User Guide. The resulting single cell transcriptome libraries contained unique sample indices for each sample. The libraries were quantified on the Agilent BioAnalyzer 2100 using the High Sensitivity DNA Kit (Agilent, 5067-4626). Libraries were pooled in equimolar ratios, and the pool was quantified by qPCR using the KAPA Library Quantification Kit - Illumina/Universal (KAPA Biosystems, KK4824) in combination with the Life Technologies Viia 7 real time PCR instrument. After the initial sequencing run, libraries were re-pooled according to estimated captured cells as determined using the Cell Ranger software (10X Genomics). Denatured libraries were loaded onto an Illumina NextSeq-500 and sequenced using a 150-cycle High-Output Kit as follows: 26bp (Read1), 8bp (i7 index), 98bp (Read2). Read1 supplies the cell barcode and UMI, i7 the sample index, and Read2 the 3’ sequence of the transcript.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Where necessary fastq files were made using Cell Ranger [14] (version 3.1.0 or 3.0.2) mkfastq. Sequencing QC was assessed using FastQC 0.11.6 and MultiQC viewer for aggregated reports.
Cell Ranger count and aggr were used to generate aggregated count files mapped to GRCz11 (Ensembl 101), without depth normalisation.
Doublets were identified from the filtered aggregated count files using Scrublet [15] in Python version 3.6 and filtered from subsequent analyses. For the MZprox1-/- mutant and Zprox1-/- mutant datasets filtered aggregated count files were processed, sc-transform normalised, filtered and clustered (louvain) using Seurat version 2.0 [16] and 3.0 [17] respectively for R statistical software version 4.0.2. QC was evaluated before and after normalisation using plot functions in Seurat and scater 1.20.1 [18], and all thresholds and settings are described in scripting. Cluster solutions were evaluated using ClusTree [19].
Datasets used in the atlas of lymphangiogenesis were processed, filtered, merged and log-normalised using Seurat version 3.0 [17], with QC and settings as above. Merged data was clustered and normalised using CSS simspec [20] and clustering and cluster evaluation performed on this object only, as described above. For all scRNA-seq datasets cluster phenotype was determined using the expression of key markers (Supplementary Table), with the aid of CellXGene visualisation software [21]. All downstream analysis and plotting were performed using Seurat version 3.0 [17] using default settings.
For velocity analysis, loom files containing RNA velocity information were first generated from the 10X data associated with each sample using velocyto.R 0.6. Relevant sample subsets were then combined with loompy 3.0.6. From pre-computed Seurat UMAPs in scRNA-seq processing and analysis, cell barcode and associated metadata were obtained and combined with the loom files. With the combined velocity scores and cell metadata, velocity maps were plotted with velocyto.R 0.6 and overlaid on the UMAPs.
Genome_build: GRCz11
Supplementary_files_format_and_content: features, barcodes and matrices output by cellranger for each sample

Submission date

Nov 06, 2021

Last update date

Apr 27, 2023

Contact name

Tyrone Chen

E-mail(s)

[email protected]

Organization name

Monash University

Department

School of Biological Sciences