title: GSM4728860: Rodent Lung CD45+ Immune Cells; Mus musculus; RNA-Seq
library_construction_protocol: Cells were maintained at room temperature throughout our single cell sample preparation. Intact lungs were dissected from 6 mice (3 males, 3 females) and placed in FACS tube with 4 ml of 1X PBS. A gentleMACS™ Dissociator 2.01 was used to mechanically liberate lung cells and cell suspension was filtered through a 70-μm cell strainer. Cell strainer was rinsed with an additional 6 ml of 1X PBS to recover remaining cells. Tissue samples were centrifuged at 2000 rpm for 5 min and supernatant was discarded. To eliminate erythrocyte contamination in single cell suspension, 2 mL of red blood cell (RBC) lysis buffer was added for 3 min. RBC lysis buffer was diluted by adding 10 ml of PBS. The resulting cell suspension was centrifuged at 2000 rpm for 5 min and supernatant containing the medium plus enzyme was discarded. Cells were resuspended in 500 μL of PBS and 12.5 μL of Fc Block was added for 15 min to block Fc receptors before being exposed to anti-CD45-PeCy7 antibody. Following another round of centrifugation at 2000 rpm for 5 min, cell pellet was first stained with 7-AAD viability dye (5μL per 200 μL) for 7 minutes in dark and then with 1 μL of anti-CD45-PeCy7 in 200 μl of final volume for 20 min. Unbound antibodies were rinsed off by centrifuging cells at 2000 rpm for 5 min and discarding the supernatant. Cells were resuspended in 300μL of PBS + 2% BSA then filtered through a 100-μm cell strainer. 7AAD- CD45+ viable immune cells were sorted in PBS + 2% BSA. Percentage of CD45+ cells was determined by flow cytometry and varied between 92-97%. Following centrifugation at 2000 rpm for 10 min, 40,000 CD45+ single cells were resuspended in a 50 μl of PBS + 2% BSA + 1 μl of RNAse inhibitor solution. To isolate immune cells comprising healthy lung tissue, 40,000 single, viable CD45+ cells were barcoded using 10X Genomics ChromiumTM Single Cell 3′ Reagent Guidelines v3 Chemistry as per the manufacturer's protocol. Quality control and cDNA quantification was performed using DNA Tape Station D1000. Sequencing was performed over two rounds on Illumina NovaSeq 125 Cycle S2 dual lane flow cell. We recovered 12,492 cells sequenced to 91,931 reads/cell (89% sequencing saturation) with an estimated doublet rate of ~8%. Read alignment to the pre-built mm10 reference genome was performed using cellranger count (10X Genomics) (Zheng et al., 2017). Single-cell suspension of CD45+ immune cells were barcoded using 10X Genomics ChromiumTM Single Cell 3′ Reagent Guidelines v3 Chemistry as per the manufacturer's protocol. Quality control and cDNA quantification was performed using Agilent High Sensitivity DNA Kit. Sequencing was performed over two rounds on Illumina NovaSeq 125 Cycle S2 dual lane flow cell. We recovered 12,492 cells sequenced to 91,931 reads/cell (89% sequencing saturation) with an estimated doublet rate of ~8%.