Single cell RNA sequencing of murine bone marrow derived macrophages and adipose tissue macrophages from lean and obese mice

Macrophages are key to innate immunity and tissue homeostasis. Plasticity of macrophages allows rapid and highly orchestrated responses to various pathogenic and physiological challenges. Capturing the dynamic intracellular programs employed by macrophages during encounters with various stimuli presents a demanding technical challenge, and failures to fully characterize some of these responses has stalled development of disease-specific therapy. Powered by single-cell RNA sequencing (scRNA-seq), we expanded current t-distributed stochastic neighbor embedding (t-SNE) single-cell transcriptome algorithms and established a novel platform, MacSpectrum (macspec.github.io), with newly generated indexes (Macrophage Polarization Index, MPI and Macrophage Maturation Index, MMI) that allow not only macrophage subpopulation characterization under various conditions but also disease-specific signature gene set identification. Our MacSpectrum model will provide a comprehensive system to annotate macrophage function and dissect their diversified programs under sophisticated conditions in vivo, which represents a major challenge in macrophage biology. Single cell RNA sequeicing profiles of 6979 M0 BMDMs , 4736 M1 BMDMs, 6391 M2 BMDMs, 1710 lean ATMs, and 1758 obese ATMs were generated. Overall design: Bone marrow cells were collected from femur and tibia bone of mice fed on regular chow diet , and were induced for differentiation to monocytes in bone marrow derived macrophage (BMDM) growth medium (Iscove's Modified Dulbecco's Medium (IMDM) + 10% FBS + 15% L-929 cell (ATCC, CCL-1)); the BMDMs were then stimulated with 100 ng/mL LPS and 50 ng/ml IFN? for M1 activation, or 20 ng/mL IL4 and 20 ng/mL IL13 for M2 activation. Unstimulated BMDMs were collected as M0 state. CD45+ CD11b+ F4/80+ macrophages (ATM) were isolated from adipose tissue of mice fed on regular chow diet (lean) or high-fat diet (obese) using a BD Biosciences FACS Aria II cell sorter. Single cell transcriptome profiles of the BMDMs and ATMs were generated on a GemCode Single Cell Instrument (10x Genomics) using Chromium™ Single Cell 3' Reagent Kit v2 (10x Genomics) and were sequenced on an illumia HiSeq 4000.