title: 10X genomics libraries for scRNA-seq of zebrafish embryos (2dpf) treated with uncoated and oligolysine-PEG copolymer coated DNA origami wireframe nanosheets
design: 10X genomics libraries for scRNA-seq of zebrafish embryos (2dpf) treated with uncoated and oligolysine-PEG copolymer coated DNA origami wireframe nanosheets
library_construction_protocol: Embryos (48 hpf) were disassociated into a single cell suspension. Four hours post injections, around 200 Tg(fli1:EGFP) embryos were euthanized in 0.02% tricaine. The tissue was dissociated with harsh pipetting in 0.25% trypsin-EDTA (GIBCO) and 100mg/mL collagenase (Sigma-Aldrich) for 3 min at 30oC, followed by resuspension in DMEM-10% FBS. The cells were then pelleted at 700xg for 5 min, washed once in 1X PBS, filtered through a 40 μm cell strainer (MERCK) and stained with 1mM DAPI solution (Abcam). TS+ cells were FACS sorted on a FACSARIA III with BD FACSDIVA software v. 9.0.1 (BD Biosciences).  Zebrafish (D. rerio) were maintained and bred in compliance with Swedish legislation on animal welfare regulations approved by Stockholms djurförsöksetiska nämnd.  Each zebrafish embryo (Tg(fli1:EGFP)) was microinjected with 4 nl of the sample. The samples for injections were prepared at a final concentration of 100 nM in 1x PBS (effective dose estimated to around 4-6 nM). Embryos at 48 hpf were anesthetized using 0.1% tricaine (MS222), transferred onto an agarose surface, placed on their ventral side, and injected into the common cardinal vein (Duct of Cuvier). scRNAseq libraries were generated from the sorted cells using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 per manufacturer's instructions, targeting 8000 cells.  The cDNA libraries were amplified using 10X Genomics Chromium Single Cell 3ʹ Reagent Kits v3.1 with 14 PCR cycles.