GSM3674240: b2; Mus musculus; RNA-Seq
Bones (femur and tibia) were harvested and placed in Media 199 (ThermoFisher Scientific, Ref#12350039) supplemented with 2% Fetal Bovine Serum (FBS, ThermoFisher Scientific, Ref#10082147). Muscle and tendon tissue was removed and bone marrow was flushed. Niche cells from the bone marrow fraction were isolated by digestion with 1 mg/mL STEMxyme1 (Worthington, Ref#LS004106) and 1 mg/mL Dispase 1 (ThermoFisher Scientific, Ref#17105041), in Media 199 supplemented with 2% FBS for 25 min at 37°C. Niche cells from the bone fraction were isolated by gently crushing and cutting bones (including epiphysis) into small fragments and digested in the same digestion mix as the bone marrow for 25 min, at 37°C with agitation (120 rpm). After digestions, both fractions were filtered through a 70μm filter into a collection tube (Fisher Scientific, Ref#08-771-2), pooled into one sample or run separately (e.g. for bone vs. bone marrow fraction scRNA-seq analysis), and erythrocytes lysed in ACK-lysis buffer (ThermoFisher Scientific, Ref#C1430) for 5 minutes on ice. Cells were then stained in Media 199 supplemented with 2% FBS for FACS cell sorting. For flow cytometry and FACS, cells were resuspended in Media 199 supplemented with 2% FBS and stained for Ter119-APC (eBioscience, Ref#17-5921-82, clone TER-119), CD71-PECy7 (Biolegend, Ref#113812, clone RI7217), CD45-PE (eBioscience, Ref#12-0451-82, clone 30-F11), CD3-PE (Biolegend, Ref#100206, clone 17A2), B220-PE (Biolegend, Ref#103208, clone RA3-6B2), CD19-PE (Biolegend, Ref#115508, clone 6D5), Gr-1-PE (Biolegend, Ref#108408, clone RB6-8C5), and Cd11b-PE (Biolegend, Ref#101208, clone M1/70) for 30 minutes on ice. Dead cells and debris were excluded by FSC, SSC, DAPI (4',6-diamino-2-phenylindole, Life Technologies, Ref#D3571) and Calcein AM staining profiles and Calcein AM (Life Technologies, Ref#C1430). FACS and cytometry was performed on a BD FACSAria II sorter, and sorted bone marrow niche cells were collected in Media 199 supplemented with 2% FBS and 0.4% UltraPure BSA (Life Technologies, Ref#AM2616). Bone marrow stroma was enriched by sorting of live cells (7-AAD-/Calcein+) negative for erythroid (CD71/Ter119) and immune lineage markers (CD45/CD3/B220/CD19/Gr-1/CD11b). For flow cytometric analysis (e.g. Figure S1K), we used Ly6a/Sca-1 (Biolegend, Ref#108127, clone), CD31 (Pecam1) (BD Bioscience, Ref#565097), Vcam1 (Biolegend, Ref#105710), CD34 (eBioscience, Ref#11-0341-82), Cdh5/CD144 (Biolegend, Ref#138006) Single cells were encapsulated into emulsion droplets using Chromium Controller (10x Genomics). scRNA-seq libraries were constructed using Chromium Single Cell 3' v2 Reagent Kit according to the manufacturer's protocol. Briefly, post sorting sample volume was decreased and cells were examined under a microscope and counted with a hemocytometer. Cells were then loaded in each channel with a target output of ~4,000 cells. Reverse transcription and library preparation were performed on C1000 Touch Thermal cycler with 96-Deep Well Reaction Module (Bio-Rad). Amplified cDNA and final libraries were evaluated on a Agilent BioAnalyzer using a High Sensitivity DNA Kit (Agilent Technologies). Individual libraries were diluted to 4nM and pooled for sequencing. Pools were sequenced with 75 cycle run kits (26bp Read1, 8bp Index1 and 55bp Read2) on the NextSeq 500 Sequencing System (Illumina) to ~70-80% saturation level.
GEO Accession
GSM3674240