GSM8154924: HAdV_lung; Mus musculus; RNA-Seq
At the time of euthanasia, mice were anesthetized with isoflurane, then injected with 2μg of anti-mouse CD45.2 PE (Biolegend, cat. #109807). 2 minutes later, mice were euthanized by CO2 asphyxiation. Subsequently, one or both lungs were removed, minced with dissection scissors, and digested in IMDM (Gibco) with 2 mg/mL collagenase (Sigma) and 20 U/mL DNAse-1 (Sigma) at 37C for 60-90 minutes with gentle rotation. Digested tissue solution was filtered through a 70-micron cell strainer (Fisherbrand), and red blood cells were removed by ACK lysis (Gibco). Intravascular CD45-PE labeled single-cell lung suspension was subjected to depletion using anti-PE microbeads (Miltenyi, cat. #130-408-801), then positive selection using a mouse CD3ε microbead kit (Miltenyi, cat. #130-094-973) according to the manufacturer's instructions. Cell numbers and viability were validated by Nexcelom Cellometer Auto 2000 (Nexcelom Bioscience) and AO/PI viability dye (Nexcelom Bioscience, cat. #CS2-0106) prior to preparation of single cell RNAseq library. To perform the 10x single cell V(D)J seq assay, 5000 live cells per sample were used to create GEMs by combining barcoded Single Cell 5' Gel Beads (10X Genomics, cat. #1000265). After GEM-RT, RT cleanup, full-length cDNA amplification and QC, T-cell receptor enriched libraries and 5' gene expression libraries were generated with Illumina P5 and P7 adapters. The libraries were quality controlled using Agilent High Sensitive DNA kit (Agilent, cat. #5067-4626) with Agilent 2100 Bioanalyzer (Agilient, cat. # G2939BA) and quantified by Qubit 2.0 fluorometer (Thermo, cat. #Q32886). Finally, the pooled libraries were sequenced with paired end single index configuration by  Illumina NextSeq 2000 (Illumina) at a final concentration of 750 pM.