title: Illumina NovaSeq 6000 paired end sequencing; Div-seq of axolotl pallium injury time course
design: Div-seq of axolotl pallium injury time course
library_construction_protocol: Anesthetized axolotls were injected intraperitoneally with 800 μM EdU (diluted in 1xPBS) at a dosage of 10 μl/g. FastGreen dye (Sigma-Aldrich) was added to the injection mix to aid visualization. Injected axolotls were kept out of water for a 15 min recovery period under benzocaine-soaked towels. After recovery, injected axolotls were returned to water. Following the desired pulse-chase period, axolotls were sacrificed and brains harvested. Animals were deeply anesthetized in 0.03% benzocaine and rectangular cranial skin/skull flaps above the right telencephalon hemisphere were performed using scalpels, forceps and spring scissors. A 1mm x 1mm piece of dorsolateral telencephalon was removed and the injury was always positioned between the choroid plexus and the olfactory bulb. After the injury, the cranial skin/skull flaps were restored without suture and the axolotls were returned to individual tanks. Frozen pallium dissections were dissociated for generating single-nuclei gene-expression libraries similarly to described above with some modifications (3). In brief, we prepared and precooled wash and Div-seq lysis buffers before moving tissue pieces to a precooled 1.5mL tube. 50µL of lysis buffer was added to the sample and dissociated via 2-5 short pulses with an electric grinder. The pestle of the grinder was washed with a 150µl wash buffer before centrifugation for 5 min at 500xg (4°C). Supernatant was removed and the pellet gently washed with 200µl of wash buffer before centrifugation for 5 min at 500xg (4°C). Supernatant was removed and the pellet was resuspended in 55µl of wash buffer. Resulting nuclei were then assessed (count and viability) using Trypan Blue assay, counted using the automated cell counter Countess (Thermo Fisher).EdU staining was performed immediately using Click-iT EdU Flow Cytometry assay Kit (Thermo Fisher Scientific, #C10424), 500 µl reaction buffer was added directly to the resuspension buffer (mix is made following the manufacturer's protocol), mixed well and left in RT for 30min. 3ml of wash buffer was added to the resuspended nuclei and mixed well, then nuclei were spun down for 5 min at 500xg (4°C), supernatant was removed and nuclei were resuspended in 500 µl PBS + 0.5% BSA with DAPI and FACS sorted immediately. For snRNAseq profiling, nuclei were diluted to an appropriate concentration to obtain approximately 2,000-10,000 nuclei per lane of a 10x microfluidic chip device. Single-nuclei cDNA was synthesized per manufacturer recommendations (10x Genomics v3.1) before continuing to library preparation with 25% of the total cDNA volume.