GSM8776891: hippocampus, patient 35, RNA; Homo sapiens; RNA-Seq
Leptomeninges were placed in 1mg/mL of Collagenase D (Roche cat. #51657128) dissolved in PBS and incubated at 37C for 10 minutes. Tissue was then mashed through a 70um strainer using the plunger of a 3mL syringe to achieve a single cell suspension. Cells were subsequently spun at 300g for 10 minutes and subjected to debris removal using a commercially available density gradient-based Debris Removal Solution (Miltenyi cat. #130-109-398). Postmortem brain was mechanically dissociated using a combination of mincing with a razor blade and dounce homogenization in a 15mL glass tissue homogenizer. Dissociated tissues were passed through a 70um filter and cleared of myelin using Myelin Removal Beads (Miltenyi cat. #130-096-433) and the MultiMacs Cell24 Separator. After debris removal, leptomeninges and brain cells were pelleted at 300xg for 10 minutes and incubated with anti-CD11b APC/Cy7 (BioLegend cat. #101226) and anti-CD45 BV510 (BD Biosciences cat. #563204) antibodies at 1:100 dilution in staining buffer (2% FBS in PBS) for 20 minutes. Cells were pelleted and resuspended in staining buffer containing propidium iodide (PI) at a 1:1000 dilution (Sigma Aldrich Cat. #P4864). CD45+/PI- cells were sorted on the Sony MA900 (Sony Biotechnology, USA) into the A1 well of a 96 well plate (Eppendorff cat. #951020401) pre-coated with 2% FBS. All sorting was performed using a 100um nozzle. Cell capture, amplification, and library construction was performed on the 10x Chromium platform using the manufacturer's publicly available protocol. Briefly, after sorting a fraction of cell suspension was used to estimate total cell count and viability. All remaining sorted cells were loaded in the Chromium platform using the 10x Genomics Next GEM Single Cell 5' v2 for scRNA and TCR-seq. Libraries were prepared using 10x library reagents in accordance with their 5' RNA and TCR sequencing library protocol and sequenced on the NovaSeq 6000.