Genetically perturbed myelin as a risk factor for neuroinflammation-driven axon degeneration

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Around 15,000 CD45lowSiglec-H+ and CD45-O1+ single cells were sorted per sample using a FACSAria III (BD Biosciences) before being encapsulated into droplets with the Chromium Controller (10x Genomics) and processed according to the manufacturer’s specifications. Briefly, every transcript captured in all the cells encapsulated with a bead was uniquely barcoded using a combination of a 16-base pair (bp) 10x barcode and a 10-bp unique molecular identifier (UMI). Complementary DNA libraries ready for sequencing on Illumina platforms were generated using the Chromium Single Cell 3′ Library & Gel Bead Kit v2 (10x Genomics) according to the detailed protocol provided by the manufacturer. Libraries were quantified by Qubit 3.0 Fluorometer (Thermo Fisher Scientific) and quality was checked using a 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent Technologies). Libraries were pooled and sequenced with a NovaSeq 6000 platform (S1 Cartridge; Illumina) in paired-end mode to reach a mean of 44,131 reads per single cell. A total of 13,556, 9,955, and 12,191 cells were captured and a median gene number per cell of 2,268, 2,562, and 2,165 could be retrieved for adult Wt, PLPmut, and PLPtg cells, respectively.