title: GSM8835550: Single cell 2-3-month WT sciatic nerves rep 3; Mus musculus; RNA-Seq
library_construction_protocol: Single Cells For single cell RNA-sequencing, sciatic nerves from 2 mice were pooled. Mice were euthanized and perfused with ice cold PBS. Nerves were collected and immediately kept on ice in DMEM with 1% FBS, RNase-In RNase inhibitor (Promega N2518), and SUPERase-In RNase Inhibitor (Invitrogen AM2696) at 1:1000 ratio. The nerves were mechanically dissociated with shears and incubated in DMEM with 1% FBS containing collagenase II (4 mg/mL; Worthington Biochemical LS004176) and Dispase II (2 mg/mL; Sigma-Aldrich D4693-1G) at 37˚C for 30 minutes with gentle shaking. The samples were pipetted up and down at 15 minutes. The cells were filtered through pre-wet 70-µm cell strainers and washed twice with buffer containing DMEM, 1% FBS, RNase-In, and SUPERase-In. Myelin was removed using Myelin Removal Beads II (Miltenyi Biotec 130-096-731) and LS columns (Miltenyi Biotec 130-042-401) per manufacturer's manuals (Miltenyi Biotec IM0001691). Additional debris and dead cells from the single cell suspensions were excluded by immediate FACS using propidium iodide (PI). Cells were collected into PBS with 10% FBS prior to library preparation.  Single Nuclei For single nucleus RNA-sequencing, sciatic nerves from 2 mice were pooled. Mice were euthanized and perfused with ice cold PBS. The nerves were collected and immediately fresh frozen in dry ice. The nerves were mechanically dissociated with shears in cold lysis buffer containing 10mM Tris buffer, 5mM CaCl2, 3mM Mg(Ac)2, 0.05% CHAPS, 320mM sucrose, 1mM DTT, 5X EDTA-free protease inhibitor (Millipore Sigma 11873580001), RNase-In RNase inhibitor (Promega N2518), and SUPERase-In RNase Inhibitor (Invitrogen AM2696) at 1:1000 ratio in nuclease-free water. The samples were transferred into a glass Dounce homogenizer on ice and mechanically dounced with 10 loose strokes and 20 tight strokes. The samples were filtered through a pre-wet 40-µm cell strainer (Fisher Scientific MT4040) and washed twice with cold wash buffer containing PBS, 0.1% BSA, RNase-In, and SUPERase-In. After removing the supernatant, the nuclear pellet was washed with 3mL of cold wash buffer. The samples were further purified using Anti-Nucleus MicroBeads (Miltenyi Biotec 130-132-997) per manufacturer's manuals.  CD45+ Single Cells For CD45+ scRNA-seq, sciatic nerves from 5-9 mice were pooled per genotype for one replicate. The samples were processed using the same procedure described above until the myelin removal step. After myelin removal, cells were incubated in CD45+ antibody (Invitrogen MCD4528; 1:250) for 20 minutes and FAC sorted using highly modified Beckman Coulter MoFlo at the Siteman Flow Cytometry core at Siteman Cancer Center. Additional debris and dead cells from the single cell suspensions were excluded during FAC sorting using propidium iodide (PI) as well. Cells were collected into PBS with 10% FBS prior to library preparation. Single Cells (All and CD45+) The purified single cells were used to prepare scRNA-seq libraries following the 10X Genomics manufacturer's manuals at the Genome Technology Access Center (GTAC) @ McDonnell Genome Institute (MGI) using 10X Genomics v3.1 chemistry.  Single Nuclei The purified single nuclei were used to prepare scRNA-seq libraries following the 10X Genomics manufacturer's manuals at the Genome Technology Access Center (GTAC) @ McDonnell Genome Institute (MGI) using 10x Genomics GEM-X v3.4 chemistry.  All libraries were sequenced using Illumina NovaSeq 6000/X accordingly to the manufacturer's manuals at the GTAC @ MGI at Washington University in St. Louis.