GSM8200516: EAE-chronic-Brain CD45-, scRNAseq; Mus musculus; RNA-Seq
Brain tissue was minced and centrifuged at 300g for 5 min at 4°C. The supernatant was discarded. Add enzyme I mixture, resuspend and then add enzyme II mixture, incubate at 37°C, 90 rpm, 3 times for 10 min each time, filter through a 70 μm filter. Then centrifuge and discard the supernatant, add pre-cooled D-PBS and resuspend, and afterwards add debris remover, mix well and slowly add pre-cooled D-PBS, thoroughly remove the upper two layers, add pre-cooled D-PBS, resuspended and centrifuge for 10 min. Centrifuge for 10 min, completely remove the upper two layers, add pre-cooled D-PBS and resuspend, centrifuge and discard the supernatant, and resuspend the cells in PBS containing 0.1% BSA and pass through a 30 μm sterile cell strainer. Library was performd accordiing to the manufacter's instructions（10×Genomics Chromium Single Cell 3' Reagent Guidelines v3 Chemistry; https://support.10×genomics.com/single-cell-gene-expression）.Cells were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion（GEM）.The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an llumina R1 primer sequence, Unique Molecular Identifier（UMI）and the 10× Barcode. The pooled barcode cDNA was then cleaned up with Silane DynaBeads, amplifiled by PCR and the apropiated sized fragments were selected for subsequent library construction. Read 2 is used for downstream analysis in Cell ranger, which is added during library construction by end repair, adenine tailing, junction ligation and PCR.