title: GSM7992592: creb3l2-full-me (40pg); Danio rerio; RNA-Seq
library_construction_protocol: At desired developmental stages (8hpf or 12hpf), approximately 30 embryos per sample were de-chonrionated with pronase. For libraries prepared with 10x chromium Single Cell 3' V3 chemistry, embryos for each condition were transferred into a 2ml eppendorf tube containing ice-cold deyolking buffer buffer (55 mM NaCl, 1.8 mM KCl, 1.25 mM NaHCO3). Embryos were pipetted up and down gently with a p1000 tip then shook at 1200 rpm at 4°C for 20 sec in a Thermal shaker. The tubes were then spun down at 4 °C with 250 g for 4 min. Supernatant was discarded and 1 ml wash buffer (10 mM Tris pH 8.5, 110 mM NaCl, 3.5 mM KCl, 2.7 mM CaCl2) was used to resuspend the cell pellet. The tubes were centrifuged again at 4 °C with 250 g for 4 min. 1 ml DMEM (gibco, 11594426) with 0.1% BSA was used to resuspend cells. Centrifuging and DMEM resuspension was repeated twice more, with the last resuspension step using 300 – 500 μl DMEM with 0.05% BSA. Cell suspension was then passed through a 40 μm cell strainer. For libraries prepared with 10x chromium Single Cell 3' V3.1 chemistry, several modifications were made to the dissociation protocol described above. Briefly, after being transferred to 2ml Eppendorf tubes, embryos were first incubated in a protease solution (10 mg/ml BI protease [Sigma, P5380], 125 U/ml DNaseI, 2.5 mM EDTA in DPBS) on ice for 4min. Embryos were then washed with a stop solution (30% FBS, 0.875 mM CaCl2 in DPBS) and gently triturated (by pipetting) in Ringer's solution. Centrifuging, resuspending, and filtering steps were then performed as in the previous protocol, but with the Ringer's solution used as the wash buffer. Cells were encapsulted and libraries were constructed with 10X Chromium instrument with the Single Cell 3' Reagent Kits (V3 and V3.1)