title: GSM7720759: zebrafish larva, neurons and glia, scRNAseq; Danio rerio; RNA-Seq
library_construction_protocol: About 150 7-day old larva were euthanized in 0.02% tricaine, and individually decapitated behind the hindbrain. They were incubated with 20 mg/ml collagenase (Life Sciences) in a buffer containing 134 mM NaCl, 2.9 mM KCl, 1.2 mM MgCl2, 2.1 mM CaCl2, and 10 mM Na-HEPES (pH 7.8) at 28 °C for 2 hr, with intermittent trituration using a p200 pipette aid at 0, 0.5 hr and 1 hr of the incubation. To release spinal cords from remaining tissue, the final triturations were done using fire-polished Pasteur pipettes with decreased opening sizes (300, 200, 100 μm respectively). Intact spinal cords were transferred to L15 media and washed 3 times with fresh media. The spinal cords were incubated with 0.25% trypsin solution (in 1xPBS containing 1 mM EDTA) at 28 °C for 25 min. The digestion was terminated by adding 500 μl stop solution (L15 with 1% fetal bovine serum). The tissue was collected by spinning at 400 g for 3 min at 4°C, washed once with L15 and resuspended in 200 μl of L15 media. Spinal cord cells were dissociated by triturating the digested tissue with fire-polished Pasteur pipettes with 80-100 μm opening. The solution was filtered through a 35 μm strainer into a siliconized collection tube. The suspension was examined on a microscope for cell count, Trypan blue staining based viability test and proportion of dispersed single cells. Samples with a viability above 70% were used for sequencing Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics).