Automated cell counter system

THP-1 cells were stimulated as described above or left unstimulated. At the end of the stimulation, cells were collected by centrifugation at 300g for 5 minutes. Cells were resuspended in 100μl of staining buffer containing 5μl of FcX blocking reagent and were placed on ice for 10 minutes. Next, 100μl of staining buffer containing CITE-seq antibodies (0.5μg/antibody/sample) was added to the cells. The cells were placed in the 4C fridge for 30 minutes to allow for antibodies to bind to their target protein. For the CITE-seq experiment antibodies were conjugated in-house following the hyper Oligo-antibody conjugation protocol as detailed here (https://cite-seq.com/protocol/). To keep track of the experimental condition (stimulated vs unstimulated) and be able to detect and remove cell doublets, cells were aliquoted into three tubes containing a uniquely barcoded hashing antibody. Cells were placed in the fridge for an additional 20 minutes. After staining was complete, all samples were washed three times with 1ml staining buffer to remove all the excess unbound antibodies. Next, cells were resuspended in 200–300μl of 1X PBS and counted using the Countess II Automated cell counter system. Immediately before loading to the 10x Genomics instrument, cells from all experimental conditions were pooled at the appropriate concentration (recovery of 10,000 cells per lane).