Illumina HiSeq 2500 sequencing; Transcriptional profiles of human circulating innate lymphoid cells and CD4 T helper cells
Transcriptional profiles of human circulating innate lymphoid cells and CD4 T helper cells
Cell Preparation Buffy coats were obtained from healthy donors at the local Blood Transfusion Center, Lausanne, Switzerland. Peripheral blood mononuclear cells (PBMCs) were isolated by density-gradient centrifugation and immediately used. Flow Cytometry analysis and cell sorting Isolated PBMCs were stained in sorting buffer (PBS, 50 μM EDTA, 0.2% BSA) with the following specific lineage marker Abs: anti-human CD8 (MEM- 31, Immunotools), anti-human CD14 (RMO52, BC), anti-human CD15 (80H5, BC), anti-human CD16 (3G8, BC), anti-human CD19 (J3-119, BC), anti-human CD20 (2H7, Biolegend), anti-human CD33 (HIM3-4, Biolegend), anti-human CD34 (561, Biolegend), anti-human CD203c (E-NPP3) (NP4D6, Biolegend), anti-human FcεRIα (AER-37, Biolegend). Additionally, we used Brilliant Violet 421 anti-human CD127 (IL-7Rα) (A019D5, Biolegend), Brilliant Violet 605 anti-human CD3 (OKT3, Biolegend), Alexa Fluor 700 anti-human CD4 (RPA-T4, Biolegend), PerCPCy5.5 anti-human CD56 (HCD56, Biolegend), Brilliant Violet 785 anti-human CD45RO (UCHL1, Biolegend), PECy7 anti-human CXCR3 (1C6, Biolegend), APC/Fire 750 anti-human CD117 (c-kit) (104D2, Biolegend),  PE anti-human CRTH2 (BM16, Biolegend), APC anti-human CD196 (CCR6) (G034E3, Biolegend). In addition, cells were stained with the LIVE/DEAD Fixable Green Dead Cell Stain Kit (Life Technologies, Grand Island, NY). Total ILCs were sorted as CD3-CD4-Lineage-CD127+, total CD4 T helper cells as CD3+CD4+CD45RO+. ILC subsets for mRNA sequencing and quantitative real time PCR (qPCR) experiments were sorted based on the expression of cKit, CD56 and CRTH2 surface markers. More specifically, ILC1s were identified as CRTH2-cKit-CD56-, ILC2s as CRTH2+cKit+/- and ILCPs as the cKit+CRTH2-. CD4 T helper subsets for mRNA sequencing and qPCR experiments were sorted based on the expression of CXCR3, CRTH2 and CCR6. Th1s were identified as CXCR3+CRTH2-CCR6-, Th2s as CRTH2+CXCR3- and Th17s as CXCR3-CRTH2-CCR6+. Cells were sorted using the FACSAria Fusion cell sorter (BD Bioscience) or the MoFlo Astrios cell sorter (Beckamn Coulter). RNeasy Micro Kit (Qiagen) SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Clontech)