title: GSM7159481: MOE, replicate 1, snATACseq; Mus musculus; ATAC-seq
library_construction_protocol: Nuclei must be purified under RNA clean conditions. A cell suspension of mouse MOE was obtained from an adult mouse following the dissociation conditions described in Monahan et al. 2017. Cells pellet were immediately resuspended in 300-uL of cold RNAse free lysis buffer (10 mM Tris-HCl, pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.1% IGEPAL CA-630), and nuclei were pelleted in a the Eppendorf 5810R Centrifuge at 1000-g, for 10-min at 4-˚C. Nuclei were resuspended in 500-uL of Homogenization Buffer (10x HB: 100-mM Trizma Base, 800 mM KCl, 100 mM EDTA, 10 mM spermidine trihydrochloride, 10 mM spermidine tetrahydrochloride in ddH2O, pH to 9-9.4 with NaOH, instructions for preparation of 1x HB can be found in Zhang et al. 2-12 50) with RNAse inhibitor (NEB MO314L), followed by 500-uL 82% Optiprep solution (4.1-mL Optiprep solution (Sigma Aldrich D1556-250ML), 25-uL 1M CaCl2, 15-uL 1M MgAc, 50-uL 1M Tris pH 8, 810-uL Water) then placed on ice. The 1-mL homogenate was carefully added onto 1-mL of 48% Optiprep solution (2.4-mL Optiprep solution, 800-uL 1M Sucrose, 25-uL 1M CaCl2, 15-uL 1M MgAc, 50-uL 1M Tris pH 8, 1710-uL Water), and was spun down in a pre-cooled swinging bucket centrifuge (Eppendorf 5810R) at 3200-g, for 20-min, at 4-˚C, acceleration 5/9, deceleration 0/9 (no break). The supernatant was aspirated and disposed, without dislodging the pellet. Air dry pellet and resuspend in 500-μL 1 x PBS diluted with 0.04% BSA with RNAse inhibitor. Cell concentration was measured for accurate loading into the 10x pipeline. Joint scRNA- and scATAC-seq libraries were prepared in collaboration with the Columbia Genome Center using the 10x Genomics Single Cell Multiome ATAC + Gene Expression kit according to manufacturer's instructions. Both GEX and ATAC libraries were sequenced to ~350M reads on an Illumina NextSeq550 100PE and Illumina NovaSeq 6000150PE, respectively.