GSM8895169: Spinal cord injury site, 3, 7, 14 days after PDGF-BB administration (10, 14, 21 d after injury) and uninjured control 2; Mus musculus; RNA-Seq
Tissues were chopped on a glass petri dish maintained on ice and transferred to a 2 ml sterile tube with 1 ml of fixation buffer and stored at 4°C for 20 hours. Samples were then processed for long-term storage at -80°C according to 10X Genomics instructions. After completing sample collection, the samples were thawed at RT and incubated at 37°C in a dissociation solution (2 ml/sample containing 0.4 mg/ml liberase) for 1 hour in agitation. Tissues were then dissociated using a glass pipette (~30 strokes/sample). Dissociated tissues were passed through a 30 μm cell strainer to remove debris. Samples were further dissociated (~10 strokes/sample) by adding 1 ml of PBS to the remaining tissue pellet. After passing all cell suspension on the 30 μm cell strainer, cells were centrifuged at 850 rcf for 5 minutes at RT. After removing the supernatant, cells were resuspended in 1 ml chilled quenching buffer. Cell suspensions were mixed with propidium iodide dye and counted (Countess 3 FL; Invitrogen). Single cell libraries were prepared using Chromium next GEM Fixed RNA Profiling Kits for Multiplexed Samples (10X Genomics). Barcoded probe hybridization was carried out with up to 2,000,000 cells per sample. Customized probes for eGFP spike-in were included during the hybridization step based on the technical note provided by 10X Genomics (Technical Note # CG000621). Each single cell cDNA library was prepared for a target of 10,000 post-probe hybridization cells per sample. Cells and 10X Genomics Chromium 3' Flex Next-GEM reagents were loaded onto chips to capture cells using a 10X Genomics Chromium iX Controller. Library preparation was according to manufacturer's protocols.