GSM9063895: Lung, WT, HDM; Mus musculus; RNA-Seq
Mice lungs were perfused by cardiac perfusion with 5 mL HBSS/1mM EDTA. Five lung lobes/mouse were harvested, manually minced with scissors, and then enzymatically digested in 2 mL of digestion buffer containing 500 μL Collagenase IA (Sigma, Cat# C9891), 100 μL DNAse I Type IV (Sigma, Cat# D5025), 250 uL Penicillin-Streptomycin (Sigma, Cat# P4333), and 5% FCS. RBCs were lysed using ACK buffer (0.5 mL, 1 minute on ice) and the single-cell suspensions from n = 3-4 mice/group were pooled together. The cells were resuspended in PBS/2% FCS, stained with an Annexin V-PE antibody (BioLegend, Cat# 640908) and DAPI (Thermo Fisher Scientific, Cat# 62248) according to the manufacturer's instructions, and sorted on a BD FACSAria cell sorter (BD Biosciences) to remove transcriptional noise from dead/dying cells. Live single cells (Annexin V and DAPI double negative cells, purity >98%) were counted, resuspended in PBS/0.04% BSA (Thermo Fisher Scientific, Cat# AM2616) in 1.5 mL RNA/DNA LoBind Eppendorf tubes (Thermo Fisher Scientific, Cat# 13-698-791) Library was constructed using the manufacter's instruction (Single Cell 3' kit v3.1 Dual Index; 10X Genomics). Briefly, total 16,500 cells per sample were loaded in duplicate on 10X Chrominium platform for Gel beads-in-Emulsion eneration (GEM). The poly-A RNA encapsulated in GEM were barcoded with unique molecular index, 10x barcode, and Illumina read 1 during reverse transcription. After post GEM-RT cleanup, cDNA amplification, enzymatic fragmentation, end repair, A-tail, ligation for Illumina Read 2 sequence, sample index PCR was conducted to add Illumina I5, P5, I7, and P7 for pair-end library construction.