title: Illumina HiSeq 4000 paired end sequencing; Human dendritic cell subsets in acute skin inflammation
design: Human dendritic cell subsets in acute skin inflammation
library_name: 23971_#292_p
library_construction_protocol: Isolation of immune cells from the blood or skin of human. Human blood samples were obtained from healthy donors under local ethics approval [14/SC/0106, National Research Ethics Service (NRES)]. PBMCs were isolated using Lymphoprep (Stem Cell Technologies) gradient isolation. Skin samples were obtained from healthy donors undergoing plastic surgery. Samples were taken under good clinical practice guidance with ethical approval of the NRES Committee South Central. Subcutaneous fat was removed completely before the sample was dissected and incubated in collagenase P (Roche) overnight at 37°C. Cold 10 mM EDTA solution was added to the sample to stop the digestion. The digested tissue was passed through a 70-μm strainer, washed with EDTA solution, and harvested mononuclear cells with Lymphoprep gradient isolation before further procedures.   Antibodies, flow cytometry and cell isolation. Surface staining was performed at 4oC in FACS staining buffer (0.5% FBS in PBS). Intracellular staining was performed using Foxp3/Transcription Factor Staining Buffer Set (Invitrogen), following the manufacturer instructions. The following anti-human antibodies were used: Brilliant Violet 650™-anti-CD3 (OCT3; Biolegend), PE/Cy7-anti-CD123 (6H6; Biolegend), APC-anti-CD1a (HI149; BD Biosciences), Alexa Fluor® 700 anti-CD1c (L161; Biolegend), PerCP-anti-CD11b (DCIS1/18; Biolegend), Brilliant Violet 421™-anti-CD11c (3.9; Biolegend), Brilliant Violet 650™-anti-CD11c (3.9; Biolegend), Brilliant Violet 785™-anti-HLA-DR (L243; Biolegend), APC/Fire™ 750-anti-HLA-DR (L243; Biolegend), PE anti-BDCA-2 (201A; Biolegend), Brilliant Violet 421 anti-BDCA-2 (201A; Biolegend), PE/Dazzle 594 anti-CD86 (IT2.2; Biolegend), Brilliant Violet 650™-anti-CD1a (HI149; BD Biosciences), Brilliant Violet 605™-anti-CD83 (HB15e; Biolegend), BUV805 anti-CD5 (UCHT2; BD Biosciences), BUV805-Anti-CD4 (SK3; BD Biosciences), BUV661-Anti-CD3 (UCHT1; BD Biosciences), Brilliant Violet 421™-anti-CD80 (2D10; Biolegend), Alexa Fluor 488-anti-Axl (108724; Bio-Techne), Alexa Fluor 700-anti-Siglec-6 (767329; Bio-Techne), Brilliant Violet 785™-anti-CCR7 (G043H7; Biolegend), APC/Cy7 anti-CD14 (63D3; Biolegend), Brilliant Violet 570™-anti-CD14 (M5E2; Biolegend), live/dead aqua (Invitrogen) and live/dead violet (Invitrogen). Flow data were acquired using FACSDiva on LSRFortessa and further analyzed with FlowJo (Tree Star) software. Blister BDCA-2+ DCs were sorted with the BD FACSAria IIIu as BDCA-2+CD123hi and BDCA 2+CD123int cells. Blood BDCA-2+ DCs and T cells were isolated by magnetic-activated cell sorting (MACS) with anti-BDCA-4 and anti-CD3 MicroBeads (Miltenyi Biotec), respectively, according to the manufacturer instructions. Blood CD1c+ DCs were isolated using BDCA-1+ dendritic cell isolation kit (Miltenyi Biotec) with BDCA-4+, CD19+ and CD14+ cells depleted from the blood before CD1c separation. For ensuring higher purity of both DC subsets, the positively labeled cells underwent a second column run.  Skin Sample Collection. Healthy donors were recruited, and each bilateral 3 mm baseline full thickness punch biopsies on the flank of the lower back were given to create skin wounds. Larger biopsies (6mm) to encompass the bilateral original wounds were taken between one to four days later. The study was under ethics approval [14/SC/0106 NRES]. Suction blister technique. Saline or HDM extract was delivered to the upper arm of the volunteers by intraepidermal skin prick test and suction blister cups were applied to the sites with vacuum pressure of 200 mmHg to create blisters. Blisters were protected overnight, and the fluid was aspirated 24 hours later to collect skin-infiltrated cells and tissue fluid. The concentration of indicated cytokines was measured by multiplex array (multiplex bead array), and the cells were stained with cell surface antibodies for flow cytometric analysis of DC subsets or for bulk or single-cell RNA sequencing analysis. Human PBMCs and blister cells were stained with antibodies in PBS for 10 minutes, and each targeted cell of interest was sorted into each well of 96-well plates containing 2 ul of 0.2% triton plus 1U/ul RNaseOUT (ThermoFisher Scientific).  Single cell libraries were prepared using Smart-Seq2 protocol with minor modifications. We performed reverse transcription using Smartscribe RT enzyme, 50U/cell (Takara).  For cDNA amplification, we performed 25 PCR cycles. All the other parameters were as described in the original Smart-seq2 paper. Not all the wells were successfully profiled, and some of the wells were excluded from the data analysis after applying QC filters.
attributes.tag: Experimental Factor: single cell identifier
attributes.value: cell23971_292
attributes.tag: Experimental Factor: cell type
attributes.value: plasmacytoid dendritic cell
attributes.tag: Experimental Factor: FACS marker
attributes.value: CD123Hi BDCA2+
attributes.tag: Experimental Factor: organism part
attributes.value: skin
attributes.tag: Experimental Factor: stimulus
attributes.value: none
attributes.tag: Experimental Factor: inferred cell type
attributes.value: not available