GSM5940216: CD1 sham-ATACseq-rep1; Mus musculus; ATAC-seq
At the indicated timepoints, mice ketamine/xylazine solution (100 mg/kg and 10 mg/kg, respectively) injected intraperitoneally. The left ventricle of the heart was perfused with ice-cold PBS (Thermo Fisher Scientific) to remove blood content from the lung. Lungs were then immediately harvested and minced in 10-cm Petri dishes and then incubated in digestive solution (DMEM, 0.2 mg/ml Liberase DL, and 100 U/ml DNase I). Samples were digested at 37°C for 45 minutes. Digestive solution was inactivated with 1x DMEM (Thermo Fisher Scientific) containing 10% FBS (Thermo Fisher Scientific). Cell and tissue suspension was put through a 40-µm filter and centrifuged. Cell pellet was resuspended in red blood cell lysis buffer (BioLegend) for 90 seconds and then diluted in 3x volume of PBS. Cells were centrifuged and resuspended in 200 µl FACS buffer (1% BSA and 0.5 mM EDTA, pH 7.4 in PBS). The single-cell suspension was then incubated with anti-mouse CD45:PerCpCy5.5 (BioLegend; 1:200), anti-mouse CD326-APC (BioLegend; 1:200), and DAPI (Sigma-Aldrich; 1:1,000) antibodies for 30 minutes on ice. Samples were then FACS sorted using a BD FACS Aria II (BD Biosciences, San Jose, CA, USA). Samples were then FACS sorted using a BD FACS Aria II (BD Biosciences, San Jose, CA, USA). To isolate GFP+ lung mesenchyme, the following selection strategy was used: debris exclusion (FSC-A by SSC-A), doublet exclusion (SSC-W by SSC-H and FSC-W by FSC-H), dead cell exclusion (DAPI by tdTomato), CD45+ cell exclusion (PerCP-Cy5.5 by tdTomato), EpCAM+ cell exclusion and isolation of GFP+ cells (APC by GFP). For the Col1a1-GFP cell isolation and analysis, an additional antibody for anti-mouse CD31:PE (BioLegend; 1:200) was used to exclude endothelial cells. For RNA analysis, GFP+ cells were sorted directly into RLT lysis buffer (Qiagen, Valencia, CA, USA). For the in vitro vs in vivo RNAseq experiment, RNA quality was determined using the BioAnalyzer (Agilent, Santa Clara, California, USA). RNA samples that had RQN values > 6 were approved for library prep and sequencing. RNA libraries were prepared using approximately 100 ng of total RNA according to the manufacturer's instructions for the TruSeq Stranded mRNA Sample Prep Kit (Illumina, San Diego, CA), employing poly-A mRNA enrichment using oligo dT magnetic beads. The final adapter-modified cDNA fragments were enriched by 15 cycles of PCR using Illumina TruSeq PCR primers.  The concentration and size distribution of the completed libraries were determined using a Fragment Analyzer (Agilent, Santa Clara, CA, USA) and Qubit fluorometry (Invitrogen, Carlsbad, CA, USA). Libraries were sequenced at up to five samples per lane, following Illumina's standard protocol using the Illumina cBot and HiSeq 3000/4000 PE Cluster Kit. The flow cells were sequenced as 100 X 2 paired end reads on an Illumina HiSeq 4000 using the HiSeq Control Software HD 3.4.0.38 collection software. Base-calling was performed using Illumina's RTA version 2.7.7. Raw fastq files were aligned to the mm10 genome using Hisat2(Kim et al., 2019). Sam files were converted to Bam files using Picard SortSam. Count matrices were generated using featureCounts from the Subread package (Liao, Smyth and Shi, 2013). Count matrices were then used for differential gene expression using Deseq2 (Kim et al., 2019). The threshold for significant differential expression was an FDR ≤ 0.05 and a log2 fold-change of ≥ 1 or ≤ -1. Normalized read counts were obtained from Deseq2.