Single-cell RNA-sequencing of the small-spotted catshark (Scyliorhinuc canicula) embryonic head
Single-cell RNA-sequencing of the small-spotted catshark (Scyliorhinuc canicula) embryonic head
Small-spotted catshark eggs were opened using dissecting scissors and the whole content of the egg was placed in a glass Petri dish with sea water. Embryos were then anesthetized on ice for 10min and staged based on morphological landmarks defined by Ballard and colleagues. The head region was cut at the level of the otic capsule and placed in ice-cold sea water on ice during the whole duration of the staging and dissecting process, to reduce cell mortality and maintain osmolarity. Once all embryonic heads were dissected, they were centrifuged at 500g for 5min at 4°C and sea water was carefully removed and exchanged for clean PBS. After carefully removing the PBS, heads were incubated in pre-warmed trypsin solution at 37°C for 10min with gentle shaking. All steps after trypsin treatment were performed inside a clean bench to avoid ambient contamination. Following trypsinization, heads were dissociated by gently pipetting, resulting in a homogeneous cell suspension. Trypsin reaction was quenched by adding pre-warmed FBS. Cells were centrifuged at 500g for 5min at 4°C and the supernatant removed without disturbing the cell pellet. Cells underwent three washes in sterile-filtered ice-cold PBS supplemented with 2.5µL of RNase inhibitors at 500g for 5min at 4°C to remove any traces of trypsin and cell debris. The resulting cell suspension was filtered through a 35µm cell strainer to remove cell clumps and obtain a final single cell suspension. Single-cell RNA-sequencing was performed following the 10x Genomics protocol for the Chromium Next GEM Single Cell 3' Reagent Kits v3.1 Dual Index (10x Genomics PN-1000269). The scRNA-seq library was generated using the Chromium Next GEM Single Cell 3' Library Kit v3.1 (10x Genomics PN-1000196).