GSM6754186: CD8 T cells, LPS, rep2; Mus musculus; RNA-Seq
Single-cell lung suspensions were obtained from LPS, saline, young, or old mice as follows: first, lungs were perfused with 10 ml PBS containing 50 U/ml heparin and placed into 2 ml c-DMEM, DMEM, 500 ml supplemented with filter-sterilized 5 ml 1M HEPES buffer, 10 ml MEM nonessential amino acid solution 100x, 5 ml Penicillin-Streptomycin 100x, 660 µl 2- mercaptoethanol 50 mM, and 45 ml heat-inactivated fetal bovine serum. A single-cell suspension was obtained using enzymatic digestion. Residual erythrocytes were lysed using Gey's solution (8 mM NH4Cl, 5 mM KHCO3 in water), passed through a 40 µm strainer, and suspended in c-DMEM. Then, 1-2×107 cells were stained for cell sorting: cells were washed in 1x PBS with 5% FBS and incubated in TruStain FcX-Fc Block diluted in 1x PBS with 5% FBS for 15 min at 4°C. Fc block was removed and cells incubated in antibody cocktails (CD45, APC/Cyanine7, clone 104; CD3, FITC, clone 17A2; CD8, Pacific Blue, clone 53-6.7) diluted in 1x PBS with 5% FBS for 20 min at 4°C in the dark. After staining, cells were washed in Flow Sort Buffer (1x PBS, 5%FBS, 10mM HEPES, 35 µg/ml DNAseI, 5mM MgCl2) and passed through a 40 µm strainer. Cells were washed again in Flow Sort Buffer and stained in Propidium Iodide (Biolegend) according to manufacturer's instructions. Cells were washed in Flow Sort Buffer once more and kept on ice until sorting using a BD FACs AriaIII. After sorting, CD8 T cells were collected into Trizol and frozen at -80°C until RNA extraction. RNA was extracted using the Direct-Zol RNA Microprep kit (Zymo Research) with an in-column DNAse I treatment, and eluted in DNase/RNase-free water, following the manufacturer's instructions. The mRNA-seq library was generated following the Smart-seq2 protocol described in Picelli et al. 2014. 0.8-10 ng of RNA were used in a reverse transcription and template-switching reaction using tailed oligo-dT primers, locked nucleic acid (LNA)-containing template-switching oligos (TSOs), and betaine for cDNA synthesis. Resulting ss-cDNA was amplified (12-14 cycles) for ds-cDNA synthesis, followed by the Nextera XT sample preparation protocol to convert cDNA to library. Samples were pooled after enrichment PCR, and 50SR sequencing was conducted using a single lane of Illumina HiSeq 3000.