GSM9045861: 5 dpf hematopoietic zebrafish cells, anterior region, replicate 1; Danio rerio; RNA-Seq
Single cell suspension for FACS sorting was prepared using an optimized protocol adapted from multiple sources (Bresciani et al., 2018; Manoli and Driever, 2012; Samsa et al., 2016). All steps were performed with cooled solutions (4°C), and samples were kept on ice throughout the processing to preserve the viability of cells. Briefly, 5dpf larvae were anesthetized using balneation in embryo medium supplemented with tricaine, at a final concentration of 640 µg/ml. Larvae (between 20 and 30 whole larvae or 60-80 dissected segments per tube) were washed twice in PBS before centrifugation (300 x g for 1 min @4°C) and the supernatant was discarded. Larvae were incubated in 1 ml TrypLE medium (Life Technologies, Cat#: 12605-010) for 10 min on ice, with gentle pipetting every 3 mins, first with a P1000 pipette and subsequently with a P200 pipette. Samples were then centrifugated for 7 min at 300g, before the TrypLE was removed and the cells were resuspended in 500 µl FACSmax medium (Genlantis, Cat#: T200100). Ultimately, samples were passed through a 40 μm cell strainer moistened with FACSmax buffer onto a 35 mm cell culture dish using a syringe plunger. The cell strainer and the dish were washed with 300 µl of FACS max and the flow-through was transferred to a FACS tube and kept on ice until sorting (within the hour). Cell sorting was performed on a BD FACS AriaIII cell sorter. For all samples, gating was done on SSC-A vs. FSC-A to collect cells, then on FSC-W vs. FSC-A to keep only singlets and on APC-A to remove dead cells marked by Draq7 (Thermofisher, Cat#: D15106). Based on the 10XGenomics Chromium V3.1 guidelines, a mix of single and double positive cells from approximately 100 dissected 5dpf outcrossed Tg(cd41:eGFP) X Tg(gata2b:RFP) double-positives larvae were collected in PBS and diluted at a concentration of around 400 cells/µL in order to load ~20 000 cells per condition (anterior and posterior halves of larvae). Chromium Next GEM Single Cell 3ʹ Kit v3.1 (10XGenomics, Cat#: PN-1000269) kit was used, and library preparation was performed according to the manufacturer recommendations (17 PCR cycles were performed for final library amplification, and library quality and concentration were assessed using the BioAnalyzer and Qubit systems). Two independent biological replicates were generated. The sequencing was performed on a Novaseq6000 platform to generate 150-bp paired-end reads, and a theoretical count of 40 000 reads per cell were sequenced.