single cell RNA sequencing of murine lungs of SPAM deleter mice after alveolar macrophage depletion
single cell RNA sequencing of murine lungs of SPAM deleter mice after alveolar macrophage depletion
4 untreated (0h) or DTx treated SPAM deleter mice were sacrificed and lung single cell suspensions were created. The cells were incubated with fluorophore labeled antibodies for FACS and TotalSeq C library for 30 minutes on 4°C. 30000 CD45-, 15000 CD45+CD64+ and 15000 CD45+CD64- cells per biological replicate were FACS sorted. Cells were pooled in the same eppendorf tube, diluted into a final concentration of 2000 cells/ μl and loaded on the 10X chromium device. The DNA libraries were prepared using the GemCode Single Cell 5′ Gel Bead and Library kit, version Next GEM v2 according to the manufacturer's instructions (10x Genomics, User Guide CG000330). Size selection with SPRIselect Reagent Kit (Beckman Coulter, B23318) was used to separate amplified cDNA molecules for 5′ gene expression and cell surface protein construction. The cDNA content of pre-fragmentation and post-sample index PCR samples was analyzed using the 2100 BioAnalyzer (Agilent).  The DNA libraries were prepared using the GemCode Single Cell 5′ Gel Bead and Library kit, version Next GEM v2 according to the manufacturer's instructions (10x Genomics, User Guide CG000330). Size selection with SPRIselect Reagent Kit (Beckman Coulter, B23318) was used to separate amplified cDNA molecules for 5′ gene expression and cell surface protein construction. The cDNA content of pre-fragmentation and post-sample index PCR samples was analyzed using the 2100 BioAnalyzer (Agilent). 