GSM9081892: Monocyte derived Naive Macrophages without K21, mRNA; Homo sapiens; RNA-Seq
Incubation at 37 °C for 6 days yielded immature macrophages. On day 6, M1 immature macrophages were activated into classical M1 macrophages by adding 50 ng/ml IFN-γ (Immunotools rh-IFNγ #11343534) and 10 ng/ml LPS (Sigma-Aldrich, Lipopolysaccharides from Escherichia coli O111:B4 #L5293), or they remained without an activation stimulus. M2 immature macrophages were activated into M2a macrophages by adding 20 ng/ml IL-4 (rh IL-4 Sigma-Aldrich #SRP3093) or into M2c macrophages by adding 20 ng/ml TGFβ (R&D rhTGFβ #7754-BH/CF), respectively. The cells were further incubated until day 10 in the presence of K21 or without it as a control. On day 10, macrophages were washed with PBS and detached in the presence of 1 ml Accumax solution (Sigma-Aldrich #SLCR6580) for 5-10 min at 37 °C. For single-cell RNA-seq analysis, 1 million macrophages per condition were washed with PBS on day 10 of the experiment and then detached in the presence of 1 mL Accumax solution (Sigma-Aldrich #SLCR6580) for 5-10 min at 37 °C. Cells were washed once again with PBS, fixed in PBS: methanol mix (ratio of 1:4), and stored in a -80 freezer until subsequent use. On the day of the experiment, methanol-fixed cells were thawed on ice and rehydrated in 1X PBS for 30 minutes on ice, filtered with a 40 μm cell strainer. Cells were barcoded using MULTI-seq Lipid Modified Oligos (Sigma-Aldrich #LMO001). After cell counting, single-cell suspension samples were pooled into four separate groups (Activated macrophages, -K21; Activated macrophages, +K21; naïve macrophages, -K21; and naïve macrophages, +K21). Ten thousand cells per sample were loaded on a Chromium Controller (10× Genomics). The scRNA-seq libraries were constructed using the Chromium Single Cell 3' v3.1 Reagent Kit according to the manufacturer's guidelines (10× Genomics). cDNA libraries were uniquely sample-indexed and pooled for sequencing. A HiSeq (Illumina) sequencing run was used to sample balance on a NovaSeq 6000 SP flowcell (Illumina) targeting > 25,000 reads/cell.