GSM8781691: Bone marrow pooled; Mus musculus; RNA-Seq
Ctrl, N2ΔCx3cr1 and RbpjΔCx3cr1 spleens (n=2 per genotype) were isolated and digested on ice for 30 min. in DMEM medium supplemented with 500 U/ml Collagenase II (Worthington) and 2% FCS. Erythrocytes were removed using red blood cell lysis buffer (BioLegend). Following thorough washing, filtering and counting, single cell suspensions of equal cell numbers (one tube per spleen) were prepared. For sample multiplexing, a cell hashing-based approach was performed using oligo-tagged antibodies (TotalSeqTM-A Hashtag derived Oligos, HTO, #A1-#A6, BioLegend). Blocking was done with anti-mouse CD16/32 (TruStain fcX) and anti-mouse CD16.2, followed by HTO tagging (#A1-#A6) and anti-F4/80-BV650 staining. After thorough washing, cells were pooled and stained with Lineage-PE antibodies (CD3, CD19, B220, Ly6G, Ter119, NK1.1, CD90.2, CD49b, CD117, FcεRIα) and anti-mouse CD11b FITC, CD11c APC and Ly6C PE-Cy7. Following the exclusion of dead (DAPI+) and CD11b/CD11c double-negative cells, Lineage-negative cells were sorted using FACSAria Fusion (BD Biosciences). Collected cells were loaded on one 10x Genomics lane for downstream scRNA-seq processing and analysis. Bone marrow cells from Ctrl, N2ΔCx3cr1 and RbpjΔCx3cr1 mice (n=2 per genotype) were counted and equal numbers were taken from each mouse in separate tubes (one tube per mouse). A cell hashing-based experimental approach via oligo-tagged antibodies (TotalSeqTM –A Hashtag derived Oligos (HTO), #A1-#A6, BioLegend) was used for sample multiplexing (Table S4). After initial blocking (anti-mouse CD16/32 BV510 and anti-mouse CD16.2 antibodies) cells were incubated with individual HTOs (#A1-#A6) and with anti-mouse CD115 APC. After an extensive washing step, cells were pooled, and stained with a cocktail of anti-mouse Lineage-PE (CD90.2, CD3, CD19, B220, Ly6G, Ter119, NK1.1 Ly6A/E), anti-PE beads (Mojosort BioLegend) and Lineage-PE+ cells were depleted using QuadroMACS and LS columns (Miltenyi Biotec). Remaining Lineage-negative cells were stained with anti-mouse CD11b BV650, CD34 PE-Dazzle 594, Ly6C PE-Cy7 and CD117 APC-Cy7 and were sorted using FACSAria Fusion (BD Biosciences). Doublets and DAPI+ cells were excluded from sorted populations. Gates were set to collect four subpopulations of myeloid cells: #1 'Progenitors' (Lineage-negativeCD115+CD11blo/-CD34+CD117+), #2 'Pre-monocytes' (Lineage-negativeCD115+CD11blo/-CD34lo/-CD117lo/-Ly6Chi), #3 'Ly6Chi monocytes' (Lineage-negativeCD115+CD11b+CD34negCD117lo/-Ly6Chi), #4 'Ly6Clo cells' (Lineage-negativeCD115+CD117negLy6Clo/-). These subpopulations, #1 - #4, were mixed with the ratio: 1 : 1 : 1.5 : 2.75 to have representative subpopulations and loaded on one 10x Genomics lane for downstream processing and scRNA-seq. Library preparation for single cell mRNA-Seq analysis was performed according to the Chromium NextGEM Single Cell 3ʹ Reagent Kits v3.1 CellSurfaceProtein User Guide (Manual Part Number CG000206 Rev D; 10x Genomics). According to the protocol a given excess of cells was loaded to the 10x controller in order to reach a target number of 25,000 cells per sample. Fragment length distribution of generated libraries was monitored using 'Bioanalyzer High Sensitivity DNA Assay' (5067-4626; Agilent Technologies). Quantification of libraries was performed by use of the 'Qubit® dsDNA HS Assay Kit' (Q32854; ThermoFisher Scientific).  Sequencing run: Generated mRNA expression libraries were pooled accordingly, denatured with NaOH, and were finally diluted to 1.8pM or 2pM according to the 'Denature and Dilute Libraries Guide' (Document # 15048776 v02; Illumina). 1.3 ml of denatured pool (including 1% PhiX) was sequenced on an Illumina NextSeq550 sequencer using one high output flowcell for 75 cycles and 400 million clusters (#20024906; Illumina). HTO libraries was loaded with a molar proportion corresponding to around 5% of sequencing run capacity. Sequencing was performed according to the following settings: 28bp as sequence read 1; 56bp as sequence read 2; 8bp as index read 1; no index read 2. In total, two such sequencing runs were performed.