Single-cell RNA-seq of Xenopus laevis spinal cord during metamorphosis

We performed single-cell RNA sequencing of the Xenopus laevis spinal cord during metamorphosis, from swimming stages (NF37–38, NF47–48) to limb stages (NF54–55, NF57–58), using the 10x Genomics platform. This dataset captures the evolutionary swim-to-limb transition in a single organism and enables analysis of how spinal circuit composition changes to support aquatic versus terrestrial motor patterns. Overall design: Spinal cord tissue was dissected from 295 NF47-48, 10 NF54-55, and 5-10 NF57-58 animals and pooled into 4 (NF47-48 and NF54-55) or 6 (NF57-58) technical replicates. At NF37-38, due to the small body size, the dorsal trunk was dissected from 230 animals, removing the belly and head at hindbrain boundary, and pooled into 4 total replicates. For all stages, acquired tissue was dissociated with liberase to obtain single-cell suspension according to previously published methods, and FACS-sorted for the presence of GFP using a BD FACS Aria III Cell Sorter. All animals, except for one replicate from NF57-58 batch, were transgenic Xla.Tg(tubb2b:maptGFP)Amaya (EXRC), expressing GFP under neuron-specific tubb2b transcriptional control, enabling us to enrich neuronal cells. Cells were further sequenced and analyzed using scRNA-seq.