GSM8488947: Old-28dpi_TCR, replicate 1; Mus musculus; OTHER
To prepare the cell suspension for 5' 10X genomic single cell RNA sequencing, mice were anaesthetized (80 mg of ketamine and 10 mg of xylazine per kilogram of body weight via i.p. injection) and transcardially perfused with 20 ml of cold 1XPBS (Thermo Fisher Scientific, 14287080). 4mm spinal cord including the lesion site or the corresponding location in the naive spinal cord segment was immediately dissected. After, spinal cord tissues were processed using the Multi Tissue Dissociation Kit 1 (Miltenyi Biotec, 130-110-201) following the manufacturer's protocol. The dissociated cells were incubated with 40ul of myelin removal beads (Miltenyi Biotec, 130-096-433) to remove myelin debris following the manufacturer's protocol. After centrifugation of the final step in the myelin removal procedures, cell pellets were resuspended in 50ul 1X MojoSort Buffer (Biolegend, 480017) and 0.5ul of TruStain FcX PLUS anti-mouse CD16/32 antibody (Biolegend, 156604) was added to the cell suspension to block Fc receptors for 10 min on ice. Antibody cocktails were prepared in 50ul Brilliant Stain Buffer (Thermo Fisher Scientific, 00-4409-42). Antibodies used for sorting were: APC anti-CD45 (30-F11, 2 µg/ml, 103111, Biolegend), Brilliant Violet 711 anti-CD8a (53-6.7, 2 µg/ml, 100759, Biolegend), Brilliant Violet 605 anti-CD4 (RM4-5, 2 µg/ml, 100547, Biolegend), FITC anti-TCR β (H57-597, 5 µg/ml, 109205, Biolegend) and LIVE/DEAD™ Fixable Aqua Dead Cell Stain Kit (Thermo Fisher Scientific, L34966, 1:1000). Cells were stained with the antibody cocktails in the dark at 4°C for 20 min followed by two times washing with 1X MojoSort Buffer. Cell sorting was conducted using BD FACSAria III flow cytometer. After FACS, cells were washed twice with 1XPBS+0.04%BSA and immediately proceeded to the 10X TCR single cell RNA sequencing procedures. Library was performed according to the manufacturer's instructions (single cell 5' V2 protocol, 10x Genomics). Briefly, cells were resuspended in the master mix and loaded together with portioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Illumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, and amplified by PCR for the subsequent V(D)J library and 5ʹ gene expression (GEX) library construction. For the V(D)J library construction, full-length V(D)J segments were amplified via PCR from the full-length cDNA using primers specific to the TCR. The appropriated sized V(D)J fragments were selected with SPRIselect reagent and used for the library construction. Full length of amplified and purified cDNA was used for the 5ʹ gene expression (GEX) library construction. During the library construction Illumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample dual index were added.