title: GSM8637427: Late, EAE, replicate6, scRNAseq; Mus musculus; RNA-Seq
library_name: GSM8637427
library_construction_protocol: At early, peak, and late stage, mice were perfused with PBS, and spinal cords were collected. Spinal cord tissues were then dissociated into a single cell suspension according to the manufacturer's protocol of Adult Brain Dissociation Kit, mouse and rat (Miltenyi Biotec, 130-107-677, we did not perform the red blood cells removal step since majority of the red blood cells had been removed with PBS perfusion). For the debris removal step, majority of the samples were processed with 38% percoll except 1 Naïve sample, 3 CFA-Ctrl samples, 3 EAE early stage samples, 2 + 1 EAE peak stage samples, and 2 + 1 EAE late stage samples were processed debris removal solution (Miltenyi Biotec, 130-107-677) according to the manufacturer's protocol to enrich more OPCs. Spinal cord single EGFP+ cells were enriched with the BD FACSAria III Cell Sorter (BD Biosciences). The cells were then lysed and washed according to the demonstrated protocol, Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression Sequencing (10x Genomics, CG000365), with modifications: the cells were centrifuged for 10 min at 300 g and 4°C, resuspended in lysis buffer (containing 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.01% Tween-20, 0.01% IGEPAL (CA-630), 0.001% Digitonin, 1% BSA, 1 mM DTT, 1 U/ul RNase inhibitor) and incubated on ice for 3 min. After the incubation, wash buffer (containing 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% Tween-20, 1% BSA, 1 mM DTT, 1 U/ul RNase inhibitor) was added on top without mixing. The nuclei were centrifuged for 5 min at 500 g and 4°C. Nuclei were washed once in wash buffer followed by another wash with diluted 1x Nuclei buffer (10x Genomics, PN-1000283) containing 1% BSA, 1 mM DTT and 1 U/ul RNase inhibitor. Libraries were sequenced on an Illumina Novaseq 6000 with a 50-8-24-49 read setup for ATAC (minimum 25,000 read pairs per cell) and a 28-10-10-90 read setup for RNA (minimum 20,000 read pairs per cell). The Chromium Next GEM Single Cell Multiome ATAC + Gene Expression chemistry (10x Genomics, PN-1000283) was applied to create single cells ATAC and RNA libraries. 26 +10 EAE mice (8 +6 mice from early stage, 8 +2 mice from peak stage and 10 +2 mice from late stage), 12 +6 CFA-Ctrl mice (4 +6 mice from early stage, 4 mice from peak stage and 4 mice from late stage) were used for independent replicates.
identifiers.namespace: GEO
identifiers.id: GSM8637427_r1