Cd14 cell positive selection

Human monocytes were isolated from human buffy coats purchased from the Massachusetts General Hospital blood bank using a standard Ficoll gradient and subsequent CD14+ cell positive selection (Stemcell Technologies). Selected monocytes were cultured in ultra low- adherence flasks (Corning) for 0, 3, or 6 days with RPMI media (Invitrogen) supplemented with 10% FBS (Invitrogen) and 50 ng/mL human M-CSF (Biolegend). SeqWell analysis was performed as previously described⁵⁴ . Briefly, at the respective timepoint, cells were detached using trypsin, spun down, and counted. Approximately 12,000 cells were loaded on each array for each timepoint and condition to minimize doublet-loading. The arrays were sealed with a semi-permeable membrane prior to cell lysis and hybridization to single-cell beads. Beads were subsequently pooled for reverse transcription and whole transcriptome amplification.

Human monocytes were isolated from human buffy coats purchased from
the Massachusetts General Hospital blood bank using a standard Ficoll
gradient and subsequent CD14+ cell positive selection (Stemcell
Technologies). Selected monocytes were cultured in ultra low-adherence
flasks (Corning) for 6 days with RPMI media (Invitrogen) supplemented with
10% FBS (Invitrogen) and 50 ng/mL human GM-CSF (Biolegend) before profiling
with single-cell RNA-sequencing.