Illumina HiSeq 4000 sequencing; Transcriptome differences in HCC infiltrated CD8+ T cells in effector and exhaustion state
Transcriptome differences in HCC infiltrated CD8+ T cells in effector and exhaustion state
Fresh specimens and blood were collected from HCC patients who underwent surgery for therapeutic purposes. For isolation of TILs, tumor tissues were minced into small pieces and digested with collagenase Type II (100 U/ml in HBSS containing calcium and magnesium) and DNase I (20 μg/ml) for 30 minutes at 37°C, then prepared by using the gentleMACS Dissociator (Miltenyi Biotech) according to the manufacture's protocol. Supernatants containing TILs were filtered through 70 μm cell strainer to obtain single cell suspension, then centrifuged at 800 g, 4°C for 30 min on a gradient consisting of 80% and 40% Percoll solutions (GE Healthcare). The T cell fraction was recovered from the interface between the 80% and 40% Percoll layers. Tumor infiltrating CD8+ T cell subsets were stained with anti-CD8a antibodies and sorted with FACSAria II cell sorter (BD Biosciences). For total and naïve CD8+ T cell negative isolation, peripheral blood mononuclear cells (PBMCs) and splenocytes density gradient separated by Ficoll (GE Healthcare) were purified by negative selection through EasySep human CD8+ T Cell Enrichment Kit and EasySep human naive CD8+ T Cell Enrichment Kit (STEMCELL). Cell purity was checked by flow cytometry. RNA-Seq The total RNA input material was used to make sequence libraries using the Illumina TrueSeq RNA Sample preparation kit v2 (Illumina). The total RNA input material was used to make sequence libraries using the Illumina TrueSeq RNA Sample preparation kit v2 (Illumina).