| Construction Protocol | To obtain single-lung-cell suspensions, lungs were extensively perfused with 3 ml of HBSS (Lonza, Cat. BE10-508F) through the right ventricle, cut into small pieces with razor blades, and digested for 1 h at 37°C in HBSS containing 5% v/v of FBS (Gibco, 10270-098), 1 mg.ml-1 collagenase A (Roche, Cat. 10103586001) and 0.05 mg.ml-1 DNase I (Roche, Cat. 11284932001). After 45 min of digestion, the suspension was flushed using a 18G needle to dissociate aggregates. PBS containing 10 mM of EDTA (Merck Millipore, 1084181000) was added to stop the digestion process and the suspension was then filtered. Staining reactions were performed at 4°C in FACS buffer (PBS containing 50% v/v of Brilliant Stain Buffer (BD, Cat. 563794), 2.5 mg.ml-1 of BSA (Sigma, Cat. A7906-100G), 0.5 mg.ml-1 of sodium azide (Acros Organics, Cat. 190385000) with 2% v/v of Fc block (BD, Cat. 553142) to reduce non-specific binding. Cell sorting was performed on a FACSAriaIII (BD Biosciences) using the nozzle 85 at a rate allowing minimum 85% of efficiency. For analysis of lung CD64+ mononuclear phagocytes, cell suspensions were first enriched in mononuclear cells by harvesting cells from the 1.080:1.038 g.ml-1 interface using a density gradient (Percoll from GE Healthcare, Cat. 17089101). Lung tissue CD64-expressing cells were FACS-sorted sorted as singlet mononuclear cell-enriched CD45+ non-autofluorescent SSCloF4/80+CD11c-Ly-6CloCD64+ cells. In the first replicate experiment ('CD64+ Exp#1), AM were FACS-sorted as singlet mononuclear cell-renriched CD45+CD11c+ autoflurorescent cells and were spiked in as controls at a ratio of 1:10. The first experiment was performed at KU Leuven (Belgium), while the second (CD64+ Exp #2) was performed at the GIGA Institute (Liege University, Belgium). For analysis of lung Ly-6Clo and Ly-6Chi monocytes, lung CD45+F4/80+CD11c-Ly-6CloCD64- cells and CD45+F4/80+CD11c-Ly-6ChiCD64- cells were FACS-sorted, respectively. For each sample, an aliquot of Trypan blue-treated cells was examined under the microscope for counting, viability and aggregate assessment following FACS sorting. Viability was above 90% for all samples and no aggregate were observed. Cell preparations were centrifuged at 1,503 RCF for 4 minutes and pellets were resuspended in calcium- and magnesium-free PBS containing 0.4 mg.ml-1 of UltraPure BSA (Thermo Fisher Scientific, Cat. AM2616). Cellular suspensions were loaded on the Chromium™ Controller (10x Genomics, Pleasanton, CA, USA) in order to generate Gel Bead-In-EMulsions (GEMs). Sequencing libraries were generated by using the Chromium™ Single Cell 3' Reagent Kits v2 (10x Genomics, Pleasanton, CA, USA) following manufacturer's instructions. GEM-RT was performed in a Veriti© 96-Well Thermal Cycler (Fisher Scientific, Merelbeke, Belgium). After RT, GEMs were broken and the cDNAs were cleaned up with DynaBeads MyOne Silane Beads (Fisher Scientific, Merelbeke, Belgium). cDNAs were then amplified in a Veriti© 96-Well Thermal Cycler. According to the expected cell recovery (based on a 60% recovery of total loaded cells), number of amplification cycles was set to 14. Amplified cDNA products were cleaned up using SPRIselect Reagent kit (Beckman Coulter, CA, USA), after what purified cDNAs were quality controlled and quantified using an Agilent High Sensitivity DNA kit on a 2100 Bioanalyser (Agilent, CA, USA). Illumina's P5, P7 and Read2 primers, as well as Sample Index were then added to generate sequencing libraries following Chromium™ Single Cell 3' Reagent Kits v2 protocol. Steps were as follows: (1) enzymatic fragmentation, end repair and A-tailing, (2) Double Sided Size Selection using SPRIselect reagent, (3) adaptor ligation, (4) post ligation cleanup with SPRIselect reagent, (5) Sample index PCR (number of cycles set to 14) and (6) Double Sided Size Selection using SPRIselect reagent. The barcoded sequencing libraries were quality controlled using an Agilent High Sensitivity DNA kit on a 2100 Bioanalyser and quantified by quantitative PCR (KAPA Biosystems Library Quantification Kit for Illumina platforms). |
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