GSM8647876: Brain, Wildtype, Day 14 post-craniotomy infection; Mus musculus; RNA-Seq
Mice were euthanized at the indicated intervals using an overdose of inhaled isoflurane prior to tissue collection. The vasculature was perfused with sterile 1X PBS, whereupon the brain was collected and placed in 1X PBS supplemented with 10% FBS on ice. For brain samples, the ipsilateral hemisphere of the brain was removed and disrupted by passing through a 70 µm cell strainer and rinsed with 1X PBS. Tissues were disrupted with collagenase IV (Cat. #C9891, Sigma-Aldrich) and DNase I (Cat. #AAJ62229MC, Fisher Scientific) for 15 min at 37°C followed by the addition of FBS to inactivate enzymatic digestion, whereupon the cell suspension was subjected to Percoll density gradient centrifugation (25% Percoll (GE Heatlhcare) with 3% FBS) at 520 x g for 20 min with no brake. The upper myelin layer down to the pellet was discarded, and the pellet was resuspended in 1X PBS to obtain a single cell suspension. For galea samples, galea was dissected from infected mice and placed into sterile 1X PBS supplemented with 10% FBS on ice. This tissue was then homogenized using the blunt end of a syringe and passed through a 70 µm cell strainer and rinsed with 1X PBS to obtain a single cell suspension. Target CD45+ cell populations were enriched via FACS before submission to a core facility at UNMC for scRNA-seq. Illumina-compatible cDNA libraries were quantified with a Qubit-30 Fluorometer and assessed using a fragment analyzer before loading on a Novaseq6000 instrument at a final concentration of 300pM for generation of 75 bp pair-end reads.