GSM9016862: Aged_Uninj_rep1; Mus musculus; RNA-Seq
Spatial - On the day of tissue collection, animals were deeply anesthetized with avertin (250 mg/kg, i.p) and then transcardially perfused with ice-cold PBS. A 4mm section of the spinal cord centered at the injury site was dissected, blotted dry with kimwipe, and embedded in OCT for parasagittal cryosectioning. Tissue blocks were stored at -80 ˚C until sectioning.   Tissue permeabilization time was determined in accordance with the Visium Tissue Optimization (TO) user guide (10x Genomics CG000238 Rev E). Briefly, 10 µm thick tissue sections were placed on the TO slide. One capture area was left empty as a negative control and 2 ug bulk-extracted spinal cord RNA (Qiagen RNeasy plus kit) was added to another empty well as a positive control. Following cDNA synthesis and tissue removal, fluorescent cDNA footprints were visualized using Nikon Eclipse microscope and 12 minutes was identified as the optimal permeabilization time. Prior to sectioning, the tissue blocks and Visium Spatial Gene Expression slide were equilibrated to the cryostat chamber for 30 minutes. To correctly identify the injury epicenter, we sampled the tissue after every eighth 10 µm section on a separate slide. Myelin was stained for 5 minutes with 1% Sudan Black in propylene glycol pre-warmed to 56˚C in a water bath and washed with running tap water until the water ran clear.  A 10 µm tissue section containing toward the midline of the tissue and containing a large injury site was captured on the Spatial Gene Expression slide. These steps were repeated for a total of four biological replicates (one per capture area) on a single slide.  The full slide was transported on dry ice to a thermocycler fitted with the Visium thermocycler adapter (3000380, 10x Genomics) pre-warmed to 37˚C. After thawing on the thermocycler adapter for 1 minute, the slide was fixed in ice-cold methanol at -20˚C for 30 minutes. Myelin was again stained with 1% Sudan Black in propylene glycol for 5 minutes at room temperature. The excess staining solution was discarded, and the slide was carefully immersed 3 times in a 50 mL conical tube of ultrapure water, then 15 times each in two additional beakers with 800 mL ultrapure water. The slide was cover slipped with 85% glycerol mounting media according to the manufacturer's instruction and imaged using Nikon Eclipse inverted microscope using brightfield microscopy.  Single Cell - Each mouse was anesthetized with Avertin (250 mg/kg i.p.) prior to transcardial perfusion with a pre-oxygenated artificial cerebrospinal fluid (aCSF) solution. From each, an 8-mm section of the spinal cord centered at the injury site was then dissected and placed in ice-cold cutting solution upon extraction until the final dissection.  For every 8 mm spinal cord (approximately 400 mg ), 50μL of Enzyme P dissolved in 1.9 mL of buffer X was prepared. The solution was then pre-warmed for at least 10min in 37°C water bath. After peeling away the meninges, the parenchymal tissue was finely chopped 10-15x in horizontal and vertical directions with a razor blade, washed with 5 ml of cutting solution, and centrifuged at 300 g for 2 minutes at RT. The resulting cell pellet was processed using the Miltenyi Neural Tissue Dissociation Kit-P (cat #130–092–628) following the manufacturer's protocol to obtain a single-cell suspension. The cell suspensions were manually triturated (slowly, 10 times) using a large-opening (1,000 um diameter) fire-polished pipette. After a 10-minute incubation at 37°C, the suspensions were further triturated with medium- and then small-opening (750 um and 500 um diameter, respectively) fire-polished pipettes to create a single-cell suspension. The suspensions were then filtered through a 70 μm cell strainer into a 50 mL conical, washed with 10 ml of HBSS with Ca2+/Mg2+, and centrifuged at 4°C and 300 g for 5 minutes. The supernatant was aspirated, and the cell pellets were resuspended in 1mL ACK Lysis Buffer for 1 minute at RT, immediately followed by the addition of 5 mL HBSS with Ca2+/Mg2+, and then centrifuged at 300 g for 5 minutes at 4°C, and the supernatant was discarded. Samples were then processed using Miltenyi Myelin Removal Beads according to manufacturer guidelines. The injury site from a separate mouse was processed as above followed by Miltenyi anti-ACSA-2 magnetic beads to enrich for astrocytes (keeping the cells retained in the column and discarding the flowthrough). The resulting cell suspension was then centrifuged at 300 g for 5 minutes at 4°C, and the supernatant was discarded. One non-enriched and one astrocyte-enriched samples were pooled to generate one biological replicate. Spatial - After imaging, the spatial gene expression slide was immersed in 3X SSC at a 45˚angle to allow the coverslip to gently separate from the slide. The tissue was enzymatically permeabilized for 12 minutes as determined by the above tissue optimization experiment and cDNA synthesis and library preparation was performed following the manufacturer's guidelines (Visium Spatial Gene Expression Reagents Kits User Guide CG000160, Rev F). cDNA amplification cycle number was determined by the Cq (defined by 25% peak RFU) value obtained by qPCR.  Each spatial sequencing sample consisted of a single animal per-10X Visium V1 sequencing area. The cDNA library generated was sequenced on an Illumina NovaSeq with a SP-200 and S1 flow cell for paired-end sequencing targeting 50 million reads per sample.   Single Cell - Each single cell sequencing sample consisted of two pooled animals with one sample enriched for astrocytes. The eight biological replicates consisted of one young uninjured, one young 3 days post-injury (dpi), three middle-aged uninjured, and three middle-aged 3dpi samples. Prior to sequencing, cell suspensions were assessed for purity, concentration, and viability with AOPI Stain imaged on a Nexcelom K2 Cellometer. After confirming viability was above 80%, cells were diluted when necessary to achieve approximately 10,000 cells per-capture. ~7,000 cells were sequenced per-sample with a mean of ~95k reads per-cell and a mean of ~11k UMIs per-cell. Sample libraries were prepared with the 10X Chromium Next GEM Single Cell 3' Reagent Kits v3.1 (Dual Index) and multiplexed on the sequencer with Dual Index Kit TT (Set A) according to manufacturer's protocols. These libraries were then sequenced using paired-end sequencing on eight channels of an Illumina Novaseq X 10-billion-read flow cell with a 300-cycle kit.