Single-cell RNA-sequencing of spinocerebellar tract neuron populations in cervical and thoracic sections of mouse spinal cord

We profiled single cells from mouse spinocerebellar tract neurons (SCTNs) in cervical and thoracic sections of mouse spinal cord, as defined by retrograde projection labeling. For each sample, SCTNs were labeled by injecting CTB (Alexa555 conjugated form, 1µg/µl in PBS) into the cerebellum using NanojetII at P4 and examined at P6-P7. Labeled SCTNs were collected manually from 150-300µm transverse spinal cord, and slices were generated using a razor blade. Labelled cells were manually collected from each injection experiment and then profiled using a plate-based SMART-SCRB protocol (see Baek et al. 2019 for detailed methods) We collected four populations of spinal cord cells: 3 control populations comprising Rostral Cervical (CRC), Caudal Cervical (CCC), Rostral Thoracic (CRT), and 1 population of rostral thoracic cells from the Hocx9-/- mutant (MRT). In addition, each plate contains wells with ERCC spike-in controls only (ERCC) and wells with a control mRNA spike-in (qRNA), which contains 10 picograms of whole mouse brain mRNA. Each library was prepared using the SMART-SCRB protocol (see Baek et al. 2019 for details), and paired-end sequenced. Read 1 contains the cell barcode (nucleotides 2-9, see BCs_4x96well.csv file) and Unique Molecular Identifier (subsequent 10 nucleotides). Read 2 contains the transcript sequence, and was aligned to the mouse transcriptome (GRCm38) using the STAR aligner with default parameters. Quantification was carried out by collapsing reads with the same UMI from the same cell mapping to the same gene, thus generating transcript counts tables. The 'all_sc_data_annotation_20180724.csv' contains the cluster identities assigned to each single-cell sample. UPDATE: Prior to 2 May 2023, the design description above stated that the cell barcode was the first 8 nucleotides in Read 1. The barcodes are actually found in positions 2-9 of Read 1. This has now been corrected.