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Sample GSM7655711

Query DataSets for GSM7655711

Status

Public on May 07, 2024

Title

ExVi2Red_01_F06

Sample type

SRA

Source name

pancreas

Organism

Danio rerio

Characteristics

tissue: pancreas
age: 4 mpf
cell type: beta-cell
genotype: Tg(ins:CaMPARI)
treatment: photoconversion

Treatment protocol

The primary islets from 4 months post-fertilization Tg(ins:CaMPARI) animals were dissected from the fish after euthanasia. Each islet was placed in a well of 24-well plate with 5 ml of DPBS medium supplemented with calcium, magnesium (ThermoFisher, 14040141), along with the appropriate concentration of D-Glucose. The islets were allowed to equilibrate for 5 min. Subsequently, the entire 24-well plate was photoconverted using a 405 nm LED array (Loctite, 1167593) for 3 seconds. The LED array evenly illuminated the entire dish with an intensity of 400 mW/cm2 and was triggered using a foot pedal switch. An internal timer using an LED controller (Loctite, 1359255) was utilized for precise timing of light exposure.

Growth protocol

4 months old Tg(ins:CaMPARI) zebrafish

Extracted molecule

polyA RNA

Extraction protocol

The photoconverted islets were dissociated into single cells by incubation in TrypLE (ThermoFisher, 12563029) at 37°C in a benchtop shaker set at 450 rpm for 45 min. Following dissociation, TrypLE was inactivated with 10% FBS, and the cells pelleted by centrifugation at 500 g for 10 min at 4°C. The supernatant was carefully discarded, and the pellet re-suspended in 500 µl of HBSS (without Ca2+, Mg2+). To remove debris, the solution was passed over a 30 µm cell filter (Miltenyi Biotec, 130-041-407). To remove dead cells, calcein violet (ThermoFisher, C34858) was added at a final concentration of 1 µM and the cell suspension incubated at room temperature for 20 minutes. The single cell preparation was sorted through 100 µm nozzle. The following channels were utilized in FACS: Pacific Blue for calcein violet; FITC (Fluorescein isothiocyanate) for green fluorescence; and PE (Phycoerythrin) for red fluorescence.
For single-cell RNA-Seq, β-cells isolated from Tg(ins:CaMPARI) islets were subjected to profiling using Smart-Seq2 protocol from Takara. For this, islets were isolated, photoconverted ex vivo, dissociated and FAC-sorted as described above. β-cells were collected from two conditions: responsive β-cells displaying photoconversion at 2.5 mM; and non-responsive β-cells without photoconversion at 16.5 mM. Photoconversion was defined as a high red-to-green ratio. For FACS of each condition, index sorting was utilized, and the cells were collected in 96-well plate containing lysis buffer (2 µl of nuclease free water with 0.2% Triton-X 100 and 4 U murine RNase Inhibitor (NEB)). For each condition a total of 93 cells were collected for profiling. Preparation of cDNA library and sequencing was performed exactly as described in a previous publication (Singh et al., Scientific Reports, 2018; doi: 10.1038/s41598-018-35218-5).
Smart-Seq2 from Takara

Library strategy

RNA-Seq

Library source

transcriptomic single cell

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Raw reads in fastq format were trimmed using trim-galore with default parameters to remove adapter sequences. Trimmed reads were aligned to the zebrafish genome, GRCz10, using HISAT2 with default parameters. htseq-count was used to assign reads to exons thus eventually getting counts per gene.
For analysis, cells with more than 800 detected genes were utilized. Transcriptional profile of responsive and non-responsive were compared using ROTS with default parameters. Genes with false-discovery rate (FDR) less than 0.05 were considered as differentially expressed.
For gene ontology (GO) analysis of differentially expressed genes, FishEnrichR was utilized. As background, genes expressed in at least 5 % of the cells were used.
Assembly: GRCz10
Supplementary files format and content: csv

Submission date

Jul 20, 2023

Last update date

May 07, 2024

Contact name

Sumeet Pal Singh

E-mail(s)

sumeetpalsingh@gmail.com

Phone

+32-473-125019

Organization name

ULB