title: GSM6721067: Spinal cord EAE + Bone marrow transplant Replicate 2; Mus musculus; RNA-Seq
library_construction_protocol: Single-nuclei preparation was performed as previously described (Hahn et al., 2022) with the following modifications. The thoracolumbar section of spinal cords were first mechanically dissociated with an ice-cold razor blade in a petri dish with 1ml EZ Prep lysis buffer (Sigma, NUC-101) on ice. Transfer of tissue suspension into a 2ml glass dounce tissue grinder tube (Sigma, D8938). Petri dish was rinsed off with another 1ml EZ buffer, which was added to the grinder tube. Suspension was homogenized by hand 25 times with pestle A followed by 25 times with pestle B while incorporating a 180-degree twist. Tissue homogenate was transferred to a fresh 15ml tube on ice. The grinder tube was rinsed with 2 ml fresh lysis buffer, which was added to the 15ml tube for a total volume of 4 ml. Samples were incubated on ice for 5 minutes. Nuclei were centrifuged at 500 x g for 5 minutes at 4°C, supernatant was removed, and the pellet resuspended with 4 ml EZ lysis buffer and incubated on ice for 5 minutes. Centrifugation at 500 x g for 5 minutes at 4°C was repeated. After removing the supernatant, the pellet was resuspended with 4 ml chilled PBS and filtered through a 35-um cell strainer into a 5 ml round bottom FACS tube (Corning, 352235). Following centrifugation at 300 x g for 10 minutes at 4°C with break 3, supernatant was gently poured out leaving behind the nuclei pellet. The pellet was resuspended in 100µl FC block buffer (BD Biosciences, 553141, 1:50 in FACS buffer) and 1ul recombinant RNase inhibitor (Takara, 2313B) incubated on ice for 5min. FACS buffer consisted of PBS (Gibco, 10010049) containing 1% BSA (Thermo Fisher, BP9700100)). To 50µl of the cell suspension, we added 150µl FACS buffer with 2µl Olig2-Alexa Fluor488 (Sigma, MABN50A4), 1ul NeuN-AlexaFluor647 (Abcam, ab190565), and 1.5ul RNase inhibitor. After 30min incubation covered from light and shaking on ice, 3ml FACS buffer were added. Following centrifugation at 300 x g for 10 minutes at 4°C with break 3, supernatant was gently poured out. The pellet was resuspended in 700µl FACS buffer with 3.5ul recombinant RNase inhibitor and Hoechst dye (Thermo Fisher, H3570, 1:2000) and incubated 4min on ice covered from light. Single nuclei were sorted on a MA900 Multi-Application Cell Sorter (Sony Biotechnology) gating for NeuN negative nuclei. 100,000 single nuclei per sample were collected into 1.5 ml DNA lo-bind tubes (Eppendorf, 022431021) containing 1 ml buffer mix with 800ul PBS, 200ul UltraPure BSA (Thermo Fisher, AM2618), and 5ul RNase inhibitor. If libraries were representing two animals, samples were pooled by collecting identical numbers (2x 50,000) of nuclei into the same sample tube. Collected nuclei were centrifuged at 400 x g for 5 minutes at 4°C with break 2. Supernatant was removed leaving 40µl suspended nuclei. Nuclei were counted using a hemacytometer (Sigma, Z359629-1EA) and assessed for concentration and quality. Reagents of the Chromium Single Cell 3' GEM & Gel Bead Kit v3.1 (10X Genomics, 1000121) were thawed and prepared according to the manufacturer's protocol. Nuclei and master mix solution was adjusted to target 10,000 nuclei per sample and loaded on a standard Chromium Controller (10X Genomics, 1000204) according to manufacturer protocols. We applied 11 PCR cycles to generate cDNA. Library construction was conducted using Chromium Single Cell 3' Library Construction Kit v3 (10X Genomics, 1000121). All reaction and quality control steps were carried out according to the manufacturer's protocol and with recommended reagents, consumables, and instruments. We chose 11 PCR cycles for library generation. Quality control of cDNA and libraries was conducted using a Bioanalyzer (Agilent) at the Stanford Protein and Nucleic Acid Facility. 10X Nuc-seq, v3.1 chemistry