title: GSM8807190: CD45 positive lung cells IAPA 4 replicates; Mus musculus; RNA-Seq
library_construction_protocol: Perfused lungs were harvested in complete RPMI (RPMI 1640 (Life-technologies)) supplemented with 10% FCS, nonessential amino acids (Gibco) and HEPES (Gibco) and kept on ice until processing. Single cells were obtained after mechanical dissociated of the lungs and digestion in digestion buffer (PBS, 3% FCS, 2 mg/ml collagenase IV) for 1 h at 37 °C. After incubation, cells were passed through a 100 µm nylon cell strainer and red blood cells were lysed with a red blood cell lysis buffer (155 mM NH4Cl, 12 mM NaHCO3, 0.1 mM EDTA). Up to 4 million cells per mouse were incubated for 30 min on ice with 50 μL of staining mix in PBS containing 0.04% BSA, TruStain FcX Block (BioLegend, cat 101320), CD45-APC (30-F11), mouse CITE-Seq antibody panel and an unique mouse TotalSeq-C cell hashing antibody (BioLegend) diluted 1:1000. The CITE-Seq panel contains 192 unique oligoconjugated antibodies and isotype controls (TotalSeq™-C). To enable simultaneous processing of multiple biological replicates, we applied a cellular hashing strategy using TotalSeq-C anti-mouse hashtag antibodies specific against mouse CD45 and MHC class I (BioLegend). Briefly, three to four biological replicates (mice) were pooled and labeled with unique hashtag antibodies prior to single-cell processing. Each biological replicate received a distinct TotalSeq-C barcode, allowing downstream demultiplexing and assignment of single-cell transcriptomic and protein expression data to individual mice. FACS-sorted single-cell suspensions were resuspended at an estimated final concentration of 2000 cells/μ, with replicates pooled in equimolar ratios and loaded on a Chromium GemCode Single Cell Instrument (10× Genomics) to generate single-cell Gel beads-in-EMulsion (GEM). The cDNA libraries were prepared using the GemCode Single Cell 5′ v3 Gel Bead and Library kit, version GEM-X V3 according to the manufacturer's instructions (10x Genomics, User Guide CG000734). 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 Fragment Analyzer(Agilent). Sequencing libraries were loaded on an Illumina NovaSeq flow cell at VIB Nucleomics core on the Illumina NovaSeq 6000 platform with a read configuration of 28 cycles for Read 1, 10 cycles for i7 index, 10 cycles for i5 index, and 90 cycles for Read 2, incorporating 1% PhiX as a sequencing control, pooled in an 85:15 ratio for the combined 5′ gene expression and cell surface protein samples, respectively