Illumina HiSeq 2500 paired end sequencing; RNA-seq expression profiling of humain MAIT cells in blood and liver as compared to mainstream TCD4+ and TCD8+ cells.
RNA-seq expression profiling of humain MAIT cells in blood and liver as compared to mainstream TCD4+ and TCD8+ cells.
Paired blood and liver samples were obtained from three uveal melanoma patients that underwent surgical intervention to remove liver metastasis in the context of a clinical trial. “Healthy” liver samples correspond to fragments that were not invaded by the melanoma. To control for a potential impact of the melanoma on blood MAIT cells blood samples from three healthy volunteers were also studied. Blood mononuclear cells were isolated on a lymphoprep gradient using a Sepmate tube (StemCell technologies) followed by red blood cell lysis (eBioscience). Liver biopsies were chopped in small pieces and digested in CO2-independent medium (Gibco) 5% FCS (Gibco) supplemented with 2mg/ml collagenase I (Sigma), 25µg/ml DNAse (Roche) and 2mg/ml hyaluronidase (Sigma) for 50 min at 37°C under constant agitation to obtain liver cell suspension. Cells were then stained using CD3, CD4, TCRgd, CD161, Va7.2, CD27 and CD45RA antibodies, sorted on an ARIA-III cell sorter (Becton Dickinson) and directly frozen in RLT (Qiagen) 5% betamercaptoethanol (Quiagen). MAIT cells were defined as CD3+CD4-TCRgd-CD161hiVa7.2+, effector memory CD4 as CD3+CD4+TCRgd-CD27+CD45RA- and effector memory CD8 as CD3+CD4-TCRgd- non MAIT CD27+CD45RA-. RNA was extracted using the Single Cell RNA Purification Kit (Norgen) according to the manufacturer's instructions with an on-column DNA digestion (Norgen). RNA quality was checked using Agilent pico chip (Agilent technologies). Half of the sample was reverse transcribed and amplified using SMART-Seq v4 Ultra Low Input RNA Kit for sequencing according to the manufacturer's instructions (Takara Bio). Quality of the process was checked using a Labchip (Caliper). Libraries were prepared on 0.6 ng of cDNA with the Nextera XT DNA Library Prep Kit (Illumina) according manufacturer's recommendations. 12 cycles of PCR were set up to amplify and add barcodes to the libraries. Quality controls were performed with Qubit (Thermofisher) to quantify the libraries, and LabChip GX Touch (Perkin Elmer) to qualify fragments length.
Experimental Factor: cell type
peripheral blood mononuclear cell
Experimental Factor: organism part
blood
Experimental Factor: disease
Uveal Melanoma