GSM7412343: ST12-1; Homo sapiens; RNA-Seq
PBMC were isolated by Ficoll-Paque (Histopaque™, Sigma Aldrich, St Louis, MO) from 15 mL of whole blood as previously described1. Enriched monocytes were isolated from peripheral blood mononuclear cells (PBMC) via two methods: Magnetic bead isolation and FACS sorting. Cells were diluted to 107 cells/mL for optimal viability and taken to the Stanford Genomic Core for scRNA seq processing as per the 10X Genomics specified protocol, briefly outlined below. Cell Counting, GEM Generation and Barcoding: An aliquot of each cell suspension was stained with trypan blue. Concentration and viability were determined using the BioRad TC20 automated cell counter. Cells were counted for correct suspension volume for 5,000 cells total. Following addition of the 10X Master Mix™ (real time reagent mix, real time primer, Additive A and real time enzyme mix) to the Single Cell A Chip, cells were loaded into the same well. Single CellGel Bead suspension was added in a separate well on the chip, as well as the partitioning oil in a third separate well. The chip is covered with a gasket and loaded into the 10X Genomics Chromium™ Controller. Following the completion of the run, the GEMs are transferred into a PCR tube for amplification. Library Preparation, and Sequencing: Single-cell libraries were prepared using the 10X Genomics Chromium 3' Gene Expression Solution. The average fragment size of each completed library was determined using the Fragment Analyzer (Agilent). Final library concentration was determined using the Qubit High Sensitivity dsDNA Assay (Thermo Fisher). Libraries were normalized to a common concentration prior to being pooled and sequenced on the Illumina HiSeq-4000.