GSM3177681: Lineage- HLA-DR+ cells sorted from DC-enriched PBMCs; Homo sapiens; RNA-Seq
PBMCs from a healthy blood donor were separated by density gradient centrifugation, enriched by magnetic bead-based sorting using the pan-DC enrichment kit from Miltenyi, and stained with aqua dead fixable kit, FITC-lineage cocktail (CD3, CD14, CD16, CD19, CD20, CD56), BV785-HLA-DR, APC-BDCA2 and V450-CD11c. Live Lineage(negative) HLA-DR(positive) cells were then sorted by flow cytometry before loading into the Chromium Single Cell Controller apparatus. For the in vitro cultures, purified CD34+ human cord blood cells were thawed and expanded for 7 days, at 5% CO2 and +37°C, in α-MEM glutamax (Life Technologies), 10% FCS (Biowest), Penicillin / Streptomycin, 1mM sodium pyruvate, 50μM β-mercaptoethanol, 25ng/ml hFLT3L, 5ng/ml hIL7, 5ng/ml hTPO and 2.5ng/ml hSCF (cytokines from Peprotech or R&D). Expanded cells were then differentiated for 14 to 21 days on a mixed feeder layer of OP9 and OP9_DLL1 cells in α-MEM, 20% FCS, Penicillin / Streptomycin, 1mM sodium pyruvate, 50μM β-mercaptoethanol, 15ng/ml hFLT3L, 5ng/ml hIL7 and 2.5ng/ml hTPO, with replacement of half of the medium on day7 and 14 with medium supplemented with 2x concentrated cytokines. Cells were harvested at the end of the culture by pipetting and filtered through a 30μm nylon mesh. Live [Live dead staining(negative)] total hematopoietic [CD45(positive)] or Lineage(negative) HLA-DR(positive) cells were sorted by flow cytometry before loading into the Chromium Single Cell Controller apparatus. Cells were partitioned into nanoliter-scale Gel Bead-In-Emulsions (GEMs) with the Chromium Single Cell Controller(10X genomics), where all generated cDNA share a common 10x Barcode. Libraries were generated and sequenced from the cDNA and the 10x Barcodes are used to associate individual reads back to the individual partitions. Analysis using molecular indexing information provides an absolute digital measurement of gene expression levels. Sequencing was performed using a NextSeq500 sequencer (Illumina).
GEO Accession
GSM3177681