GSM5777135: EAE_Peak_ATAC; Mus musculus; ATAC-seq
Mice were sacrificed with a ketaminol/xylazine intraperitoneal injection followed by intracardiac perfusion with PBS where after spinal cords were collected and dissociated using the adult brain dissociation kit (130-107-677, Miltenyi) following manufacturer's instructions. Instead of myelin debris removal (Miltenyi), Percoll (Cytiva 17-0891-01) mixed with HBSS was used to generate a percoll gradient of 38% to remove myelin debris. Immediately after dissociation, cells were stained with DAPI and sorted on a FACS Aria III cell sorter (BD Biosciences). Sox10GFP+/DAPI- cells were collected in PBS + 0.5% BSA. The pool of cells was then lysed and washed according to the Demonstrated Protocol: Nuclei Isolation for Single cell ATAC Sequencing (10x Genomics) with modifications from the Demonstrated Protocol: Nuclei Isolation for Single Cell Multiome ATAC + Gene Expression Sequencing (10x Genomics)  as follows: the cells were centrifuged for 10 minutes at 300xg and 4°C, resuspended in ATAC lysis buffer (containing 0.01% IGEPAL (CA-630), 0.01% Tween-20, 0.001% Digitonin, 1% BSA, 1 mM DTT, 1 U/ul RNase inhibitor, 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) and incubated on ice for 3 minutes. After the incubation, wash buffer (containing 0.1% Tween-20, 1% BSA, 1 mM DTT, 1 U/ul RNase inhibitor, 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) was added on top without mixing and the nuclei were centrifuged for 5 min at 500xg and 4°C. Nuclei were washed once in Diluted Nuclei buffer (10x Genomics) containing 1% BSA, 1 mM DTT and 1 U/ul RNase inhibitor, and incubated for 60 minutes at 37°C in tagmentation mix (10x Genomics). The Chromium Next GEM Single Cell Multiome ATAC + Gene Expression v1 Chemistry was used to create single nuclei ATAC and RNA libraries from the same cell. Two EAE and two CFA-Ctr animals were used for independent replicates. Libraries were sequenced on an Illumina Novaseq 6000 with a 50-8-24-49 read set-up for ATAC (minimum 25 000 read pairs per cell) and a 28-10-10-90 read set-up for RNA (minimum 20 000 read pairs per cell). Chromium Next GEM Single Cell Multiome ATAC + Gene Expression