title: GSM9034875: EAE D25-scRNA-seq; Mus musculus; RNA-Seq
library_construction_protocol: EAE mice at different disease stages, including naïve (day 0), priming (day 13), peak (day18), and chronic stages (day 25, day30, and day 35), were deeply anesthetized with pentobarbital sodium and perfused transcardially with ice-cold saline. The whole spinal cord was immediately removed and washed with prechilled PBS. For scRNA-seq, three spinal cords were pooled as one sample and dissociated into single cell suspensions for sequencing. For in situ sequencing, spinal cords were embedded in Tissue-Tek OCT compound (Sakura) and snap-freezing using dry ice. The OCT-embedded spinal cords were then coronally cryosectioned to a thickness of 10 μm using a cryostat (Leica CM3050S). The lumbar sections were collected and layered onto a spatially barcoded array for the subsequent in situ sequencing. CUT&Tag was performed with CUT&Tag (Cleavage Under Targets and Tagmentation, Hyperactive pA-Tn5 Transposase for CUT&Tag, S603-01, Vazyme) method. In brief, 100,000 cells were collected and bounded to Concanavalin A–coated beads. Isolated MG/Mac were resuspended in antibody buffer and incubated with primary antibodies against H3K18la (PTM-1427RM, PTM BI) or H4K12la (PTM-1411, PTM BIO) and secondary antibodies in order. The samples were incubated with pA-Tn5 transposase. After transposon activation and tagmentation, DNA was isolated, amplified, and purified to construct library. The library for sequencing was constructed with VAHTS Turbo DNA Library Prep Kit for Illumina (Vazyme Biotech) according to manufacturer's instructions. AMPure XP beads were used for purification steps. The library was quantified with VAHTS Library Quantification Kit for Illumina (Vazyme Biotech) and sequenced on an Illumina novaseq 150PE.