title: GSM7311139: Zebrafish heart 105275; Danio rerio; RNA-Seq
library_construction_protocol: To isolate immune cells, pools of three whole ventricles from Tg(mhc2dab:EGFP); Tg(ptprc:DsRed) zebrafish (containing at least one female and one male) were used.  Ventricles were cut into small pieces and dissociated in a 1× PBS solution containing 200 µg/ml of Liberase DL (Roche) for 20 min at 33ºC with constant agitation.  The solution was gently pipetted up and down every 3 min to promote tissue disaggregation.  The cell suspensions were passed through a 35 µm nylon mesh by centrifugation into Falcon round bottom polystyrene test tube (Corning Inc.) at 300 g for 4 min at 4ºC. EGFP+ and/or DsRed+ cells were sorted using a FACSAria III (BD) sorter, following exclusion of dead cells using DAPI staining.  Cells were sorted into a 20% FBS solution (in 1× PBS) and subsequently used for scRNA-Seq analysis. Single cell RNAseq was performed on ICELL8 platform (Takara Bio) using the V2 protocol with in-chip preamplifcation. Samples were pre-sorted into HBSS (Gibco) in a concentration of 20 cells/µl and stained with Hoechst dye (Invitrogen) for 5min at RT prior loading to MSND dispenser. For each sample 1 dispense (in ~630 wells) were made on one 5184 well chip resulting in 1479 wells containing a single cell after selection in CellSelect software (Takara Bio). Selected wells were processed for RT, in-chip preamplification and cDNA was pooled for final library preparation using standard Nextera protocol (Illumina). Library was checked in Labchip Gx touch 24 (Perkin Elmer) and sequenced on Nextseq500 (Illumina) using V2 chemistry and paired-end setup (11bp Read1, 80bp Read2). A total of 656M reads were obtained.