title: GSM7605330: Bone_old_CITE; Mus musculus; OTHER
library_construction_protocol: Mice were euthanized according to standardized and approved IACUC protocols. Femurs and tibiae were isolated, cleaned of soft tissue, cut at both ends, and marrow centrifuged out of the diaphyses and metaphyses into a collection tube. Marrow was resuspended in 1mg/mL Liberase DL (Sigma) diluted in FACS buffer (0.5% BSA [Sigma] in PBS) and digested at 37°C for 30 minutes to increase yield of stromal cells released from the vasculature fraction. Diaphyses and metaphyses cleared of bone marrow were gently crushed, rinsed in PBS, and then digested in 300 Units/mL of Collagenase IA (Sigma), diluted in MEM α (ThermoFisher), 3 times for 25 minutes each. Bone and marrow solutions were then combined and treated with RBC lysis buffer (ThermoFisher) to clear erythrocytes. The sample was then depleted of cells expressing hematopoietic lineage markers (CD5, CD45R [B220], CD11b, Gr-1 [Ly-6G/C], 7-4, and Ter-119) using Magnet Assisted Cell Sorting (MACS) and the Lineage Cell Depletion Kit (Miltenyl Biotec). Samples were incubated with anti-mouse CD45-FITC at 1:400 dilution and anti-mouse CD24-TotalSeq B at 1:400 dilution in FACS buffer at 4°C for 20 minutes in the dark. Cells were then incubated with SYTOX blue at 1:4,000 for 5 minutes, spun down at 300xg for 5 minutes at 4°C, then resuspended in FACS buffer at 1x10^7 cells/mL and sorted for CD45-SyTOX- cells on a FACS Aria II (BD Biosciences). Live Lin-CD45- cells were then washed twice in 1x PBS + 0.04% BSA and immediately submitted to the Mayo Clinic Genome Analysis Core for single-cell sequencing. Cells were counted and measured for viability using the Vi-Cell XR Cell Viability Analyzer (Beckman-Coulter).  The Chromium Next GEM Single Cell 3' Library and Gel Bead Kit (10x Genomics) was used for cDNA synthesis and standard Illumina sequencing primers and a set of unique i7 Sample dual indices (10x Genomics) were added to each cDNA pool. All cDNA pools and resulting libraries were measured using Qubit High Sensitivity assays (Thermo Fisher Scientific) and Agilent Bioanalyzer High Sensitivity chips (Agilent). Pooled libraries were then sequenced on Illumina NextSeq 2000 at 40,000 and 50,000 fragment reads/sample following Illumina's standard protocol on a P2 flow cell. The P2 flow cell was sequenced at 100 x 2 paired end reads using a NextSeq P2 sequencing kit, NextSeq Control Software v1.4.1 and base-calling was analyzed using Illumina's RTA version 3.4.4.