title: Molecular Pixelation, MPX of REH cells:  DMSO control, rep 2
design: 1 million cells from each condition in duplicate were processed for single-cell spatial proteomics (Molecular Pixelation, MPX) according to the user manual of the of the Single Cell Spatial Proteomics Kit, Immunology Panel I, Human (Pixelgen Technologies. #PXGIMM001). In summary, cells were fixed with 2% methanol-free paraformaldehyde (PFA, ThermoFisher #28906), rinsed with a wash buffer, blocked, frozen and stored at -80C until further processing. The cells were then thawed and incubated with 80 antibody oligonucleotide conjugates (AOCs) which contain information about molecular targets and unique molecular identifiers (UMI). Cells with bound AOCs were then allowed to hybridize with single-stranded DNA molecules (DNA pixels A) that contain a concatemer of a UMI sequence called unique pixel identifier (UPI). DNA pixels A could incorporate the UPI-A barcode onto the oligonucleotides of neighboring AOCs by a gap-fill ligation reaction which created spatial protein neighborhoods on the cell surface. DNA pixels A were then degraded enzymatically (only the UPI-A barcode remained on the AOCs) and a second set of UPIs (UPI-B) were incorporated onto the AOCs with a second set of DNA pixels (DNA pixels B) in a similar manner to generate overlaps between spatial zones defined by UPI-A which generated a spatial proteome map. DNA pixels B were degraded enzymatically and only the UPI-B barcode remained on the AOCs. The samples were then indexed with unique primer sets and the libraries were amplified by PCR. The AMPure XP Reagent (Beckman Coulter Life Sci.) was used to purify the amplified PCR products with a magnetic separator (Parse Biosciences #SB1004).
library_name: FU-198-Reh-2-2_S8_L001