Single-cell transcriptomics of zebrafish brains

Wild type and two-timepoint edited 23–25 dpf zebrafish brains were similarly processed for inDrops single-cell transcriptome barcoding^{4 (link),53 (link)} except that two-timepoint edited zebrafish were first heat shocked for 45 min at 37 C to induce scGESTALT barcode mRNA expression. Whole brains were dissected and dissociated using the Papain Dissociation Kit (Worthington), according to the manufacturer’s instructions with the following modifications to ensure high quality cell isolation for scRNA-seq⁵⁴ . Brains were dissociated with 900 μl of 10 units/ml of papain in Neurobasal media (Life Technologies) and incubated at 34 C for 20–25 min with gentle agitation. Samples were then gently triturated with p1000 and p200 tips until large pieces of tissues were no longer visible. Dissociated cells were washed 2x with DPBS (Life Technologies) at 4 C and sequentially filtered through 35 μm (BD Falcon) and 20 μm (Sysmex) mesh filters. Cells were resuspended in 300–400 μl DPBS and counted using an automated Bio-Rad counter. Cells were then diluted to ~100,000 cells/ml in 18% optiprep/DPBS solution. Cells were loaded onto the inDrops device and encapsulated at a rate of 10,000–20,000 per hour. Transcriptomes were obtained for ~70% of cells introduced into the device.