GSM7666349: Recent Onset sample1 on Monocyte; Homo sapiens; RNA-Seq
Cryopreserved PBMC samples of 8 age-matched subjects (2 well-controlled ROT1D patients, 2 HRS, 2 LRS, 2 uHC) were thawed and incubated with the fixable Live/Dead Violet dye (Life Technologies, Grand Island, NY, USA) for 30 minutes on ice.  The cells were subsequently stained with antibodies targeting CD14 (clone M5E2; BD Biosciences, Franklin Lakes, NJ, USA), CD16 (clone 3G8; BD Biosciences), HLA-DR (clone G46-6; BD Biosciences) for 30 minutes on ice and then washed and passed through a 30µM filter.  PBMCs were sorted with a FACS Aria III cell sorter (BD Biosciences), with specific forward and side scatter settings to exclude doublets.  Using a hemocytometer and trypan blue staining, the recovered HLA-DRhi, CD14+, CD16+, and HLA-DRlow, CD14-, and CD16- cells were determined to possess >40,000 cells/sample and viability exceeding than 90%.  These two populations were mixed in a 1:1 ratio to generate a monocyte-enriched, single cell, PBMC population. Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics)