## 2 Materials and Methods
### Cell Identification  
The nomenclature classifying cell types within each identified cluster for CON samples at 2 weeks was based on a set of \"marker genes\" based on reports within the literature (see Table 1). For CON samples, we first validated the presence of known cell subsets based on prior scRNA-seq studies of intervertebral cells, endothelial cells, myeloid and lymphoid cells of the rat IVD [5; 15]. Violin plots of gene expression levels for all cells within each cluster were used to define the majority cell type in each cluster.  
As a majority of cells in the CON populations were identified with a cluster named intervertebral disc cells, we sought to further sub-cluster this population in a post-hoc process separate from the data for cells mapped to the other clusters. IVD cells for CON samples at 2 weeks were subjected to PCA to select an optimal number of principal components, followed by K-means clustering on pooled data to identify further separation of cells in the intervertebral disc clusters based on the lowest Davies-Bouldin index.  
The above-described process for clustering and sub-clustering was repeated for CON samples at 8 weeks post-surgery following the same cluster nomenclature and marker genes. We sought to retain five major cell clusters for samples from the LDP population at both 2 and 8 weeks post-surgery, in order to directly compare cell numbers and gene expression levels against those of the CON population. In large part, the nomenclature  
\begin{table}
\begin{tabular}{c c c c} \hline \hline
**Cell Type** & **CON** & **LDP** & **Gene Marker** \\ \hline \multirow{4}{*}{Intervertebral disc cells} & 2 week (87.7\%) & 2 week (82.4\%) & _Cat2x1_—collagen type II alpha 1 chain \\  & 8 week (83.7\%) & 8 week (87.4\%) & _Sex3_—SKY-box 9 \\  & & & _Fond–_fibromodulin_ \\  & & & _Comp–_cartilage oligomeric matrix protein \\ \hline \multirow{4}{*}{Endothelial cells} & 2 week (7.3\%) & 2 week (6.41\%) & _Plump_—plasmalemma vesicle-associated protein \\  & 8 week (0\%) & 8 week (2.3\%) & _Jf1 ftm3_—interferon-induced transmembrane protein 3 \\  & & & _F11—_tyrosine-protein kinase receptor \\ \hline \multirow{4}{*}{Myeloid cells} & 2 week (1.0\%) & 2 week (1.4\%) & _Cd14_—monocyte differentiation antigen CD14 \\  & 8 week (1.2\%) & 8 week (0.8\%) & _Mcr1_—monocyte receptor C-Type 1 \\  & & & _Cd68_—CD68 antigen \\  & & & _Lyz2_—lysozyme 2 \\ \hline \multirow{4}{*}{Lymphoid cells} & \multirow{2}{*}{2 week (0.2\%)} & \multirow{2}{*}{2 week (8.2\%)} & _Cat279_—B-cell antigen receptor complex-associated protein beta chain \\  & & & _Blink_—B cell linker \\  & 8 week (8.4\%) & 8 week (5.1\%) & _Pptre_–protein tyrosine phosphatase receptor type C \\  & & _Myb_—MYB proto-oncogene, transcription factor \\ \hline \hline \end{tabular}
\end{table}
Table 1: Genes examined for definition of cell subtypes in the IVD of rat samples (i.e., marker genes). Percentages are numbers of total cells assigned to each cluster averaged across four rats. CON—cells from control (unoperated) IVDs; LDP—cells from lumbar disc punctured IVDs at 2 or 8 weeks following surgery.  
for these five cell clusters did not differ from that of CON populations; however, the numbers of cells mapped to each cluster and gene expression for marker and novel genes varied substantially.