GSM8634334: LPS Day6; Homo sapiens; RNA-Seq
Three microfluidic HSEs containing primary CD14+ monocytes were generated per condition (Con Day1, LPS Day1, Con Day6, LPS Day6). For single-cell RNA sequencing (scRNA-seq), the samples were incubated with 300 μl of 50 FU / ml nattokinase (MedChem Express) dissolved in sterile PBS for 1 h at 37 °C 5 % CO2. After 1 h incubation, the fibrin gel had been digested and the cell suspensions from each microfluidic HSE were transferred into sterile 15 ml falcon tubes (Corning, Corning Inc). Samples were then centrifuged at 200 G for 15 min at 4 °C. After centrifugation, the supernatant was carefully pipetted and discarded and cell pellets from each sample were resuspended in 40 μl FACs of buffer and kept on ice.  A CD45 positive selection was performed according to the manufacturer's instructions (Miltenyi Biotec), to separate monocyte-derived cells from fibroblasts, endothelial cells and keratinocytes in the samples. The CD45 positive (monocyte-derived cells) and CD45 negative (fibroblasts, endothelial cells and keratinocytes) samples were resuspended in 500 μl DPBS (Sigma Aldrich, Missouri, USA) and filtered through 70 µm pore sterile Flowmi Cell Strainers (Merck, Sigma Aldrich, Missouri, USA) into sterile Eppendorf tubes and kept on ice.  Acridine orange and propidium iodide (lot number: AP0BBC0301, Logos Bio, Allied Genetics Inc) was used to assess the viability of single cell suspensions derived from the HSEs, before the single cell suspensions were sequenced. A viability cut-off of > 80 % was set for each sample to proceed. Staining was performed according to the manufacturer's instructions. Stained samples were pipetted onto Luna cell counting slides (lot number: L201B1D2GB, Logos Bio, Allied Genetics Inc). The viability of the single cell suspensions was determined using the automated LUNA-FX7™ Dual Fluorescence Cell Counter (Logos Bio, Allied Genetics Inc). 7500 live CD45 negative cells were combined with 7500 CD45 positive live cells, from each condition, in a sterile Eppendorf tubes and kept on ice. Single-cell RNA sequencing (scRNA-seq) was performed by the Genome Centre, Blizard Institute, Queen Mary University of London using the Chromium Next GEM Single Cell 3' assay (10X Genomics). 15,000 cells were loaded per lane (4 lanes, 15,000 cells per condition) of the Single Cell chip (10X Genomics). A total of 35,987 cells were sequenced (14,410 cells from the Control Day 1 condition, 7516 cells from the Treated Day 1 condition, 7999 cells from the Control Day 6 condition and 6062 cells from the Treated Day 6 condition). Chromium™ 10x single cell 3'v3.1 libraries were generated and library pool QC was performed using the Qubit 2.0 Fluorometer (ThermoFisher) and the Agilent 4200 Tapestation (TapeStation Analysis Software 4.1). Final libraries were run using the Illumina NextSeq2000 sequencing system to achieve a minimum depth of 50,000 raw reads per cell. NextSeq2000 QC resulted in a quality score (% bases > Q30) of 94.91 %.