Illumina NovaSeq 6000 paired end sequencing; snRNA-seq of microdissected regions from the axolotl pallium
snRNA-seq of microdissected regions from the axolotl pallium
D_1_p
Axolotls were fully anesthetized and decapitated with scissors. Brain was then microdissected and snap frozen. Frozen pallium microdissections and whole pallium dissections were dissociated for generating single-nuclei gene-expression libraries following a modified protocol from 10x (Demonstrated protocol CG000365, Rev B). In brief, we prepared and precooled wash and 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 50-100µl of PBS + 0.5% BSA. Resulting nuclei were then assessed (count and viability) using Trypan Blue assay, counted using the automated cell counter Countess (Thermo Fisher).   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.