title: GSM7993359: Tg control (stim2a;stim2b)+/+, replicate 4, scRNA-seq; Danio rerio; RNA-Seq
library_construction_protocol: 5 dpf larvae were anaesthetised by tricaine methanesulfonate (MS222), a neuromuscular blocker (Collymore et al., 2014), and heads were dissected in Dulbecco's Modified Eagle Medium/Nutrient Mixture F-12 (DMEM/F-12; with Ca2+, and L-glutamine, and without HEPES, and Phenol Red; Gibco) with 10% foetal bovine serum (FBS; Gibco). After eyes were removed promptly, tissue dissociation and single-cell suspension were made according to previously described methods (Bresciani et al., 2018) for zebrafish and placed on ice.  Immediately after isolation of single-cell suspension, the cells were sorted based on GCaMP5G fluorescence (with 488 nm excitation and 510 nm emission) to obtain the cells of neuronal origin. The GCaMP5 was expressed under the elav/Huc promoter, which is an early marker of neuronal cells (Kim et al., 1996, Feng et al., 2019). Cell sorting was performed with a BD FACSAria II (BD Biosciences, Franklin Lakes, NJ) with support from the Core Facility at the International Institute of Molecular and Cell Biology in Warsaw. Cell viability was measured using the trypan blue staining method, and when the viability of cells was ~80%, the cells were immediately loaded on the 10X Chromium system for droplet encapsulation as described previously (Zheng et al., 2016). Approximately 8000 cells were sorted and suspended in ~40 μl volume. Cells were loaded according to the Chromium single cell 3′ kit's standard protocol (V3 chemistry). To prepare the cells for droplet-based sequencing, GCaMP5G-positive single-cell suspension was carefully mixed with reverse transcription mix before loading the cells on the 10X Genomics Chromium system, the standard manufacturer's protocol. During the encapsulation, the cells were lysed within the droplet, and they released polyadenylated RNA bound to the barcoded bead, which was encapsulated with the cell. Following the 10x Genomics user manual's guidelines, the droplets were directly subjected to reverse transcription, the emulsion was broken, and cDNA was purified using Silane beads. After the amplification of cDNA with 13 cycles, purification and quantification were performed. The 10X Genomics single-cell RNA-sequencing library preparation – involving fragmentation, dA-tailing, adapter ligation, and 12-cycle indexing PCR – was performed. After quantification, the libraries were sequenced at the Nencki Institute of Experimental Biology on an Illumina NextSeq 550 machine using a HighOutput flowcell in paired-end mode (R1: 26 cycles; I1: 8 cycles; R2: 57 cycles), thus generating 80-125 million fragments.