title: GSM8164410: LPS induced, vehicle treated, day 4, biological replicate 1-4, scRNAseq; Mus musculus; RNA-Seq
library_construction_protocol: For isolation of single cell suspensions from lung, mice were euthanized followed by whole body perfusion via the injection of 15 mL ice cold PBS through the right ventricle. Upon lung whitening at the end of the perfusion period, the trachea was then exposed, and a small nick was made on the ventral surface. 1 mL of 10 U/mL Dispase diluted in PBS was slowly injected into the lung via the trachea followed by 0.6 mL of lukewarm 1% low-melt agarose. Lung was then covered with ice for 2 min to allow the agarose to solidify. The lung was then excised and then transferred to media containing 5 U/mL Dispase, 1 mM EDTA (Gibco), and 0.1 mg/mL DNase1 in DMEM followed by incubation at room temperature for 45 minutes. The lung was then minced with a razor blade and the resulting cell suspension was filtered through a 70 µm cell strainer followed by a 40 µm cell strainer. Red blood cells were then lysed using RBC lysis buffer and the cells were again filtered through 40 µm cell strainer. After the last centrifugation step at 300 g for 5 minutes, single cells were triturated in 5 mL of PBS containing 10 µM EDTA, counted, and cellular separation and overall cell viability was assessed (overall above 90%). Final single cell suspensions containing 1,000 cells/μL were typically achieved and used immediately in single-cell RNA sequencing-based experiments. Cells pooled from four samples were loaded on a 10X Chromium Controller using Chromium Next GEM Single Cell 3ʹ v3.1 reagents (10X Genomics) with a targeted cell recovery of 30,000 cells per reaction or 7,500 cells per sample. Single-cell RNAseq libraries were prepared per the manufacturer's instructions. Single-cell RNA libraries were analyzed and quantified using a Bioanalyzer 2100 system (Agilent) and Qubit HS DNA quantification kit (ThermoFisher). Libraries were pooled equimolarly and quality-controlled under a 1 million-read sequencing using MiSeq system (Illumina). Single cell gene expression libraries and cell multiplexing libraries were then sequenced on a NovaSeq 6000 with S4 flow cell ((Illumina) using paired-end, dual indexing sequencing with 28 cycles for read 1 and 90 cycles for read 2. Single cell gene expression matrices were generated with an average of ~6,000 cells assigned per sample, and ~1,900 genes detected per cell.