GSM8461040: JHOS2 cells, HTO; Homo sapiens; OTHER
JHOS2 cells were harvested according to standard cell culture procedures. After medium removal, the cells were washed with 6 ml of phosphate-buffered saline (PBS) and detached with 1 ml of TrypLE Express Enzyme (#12605010, Gibco™, Thermo Fisher Scientific) incubation for 10–15 minutes at 37°C and 5.0% CO2 before quenching with 5 ml of warm medium. During incubation, the pre-printed drugs in the 96-well plates were dissolved in 25 µl of medium while shaking for 15 minutes. The cell suspension was then transferred to a 15 ml tube and centrifuged at 300 g for 5 minutes. After removing the supernatant, the pellet was resuspended in 5 ml of warm medium before cell counting, which was performed as described before. The objective was to plate ~13,000 cells per well by adding 75 µl of the cell suspension to each well after dissolving the drugs, to obtain a final volume of 100 µl per well of each 96-well plate. Finally, cells were incubated for 24 hours at 37°C and 5.0% CO2 before harvesting for Cell Hashing. Live-cell labeling was performed as previously described by Stoeckius et al. (2017, DOI: 10.1186/s13059-018-1603-1) with pairwise combinations of 20 in-house produced antibody-oligo conjugates directed against β2microglobulin (#316302, BioLegend, San Diego, CA, USA), and CD298 (#341712, BioLegend). Single-cell gene expression profiles were studied using 10x Genomics Chromium Single Cell 3'RNAseq platform (10x Genomics, Pleasanton, CA, USA). The Chromium Single Cell 3'RNAseq run and library preparation were done using the Chromium Next GEM Single Cell 3' Gene Expression version 3.1 Dual Index chemistry with Feature Barcoding technology (see user guide #CG000317, 10x Genomics) The Cell Hashing protocol described by Stoeckius et al. (2017, DOI: 10.1186/s13059-018-1603-1) was used to generate the HTO/Antibody Capture library and a 3' mRNA library. https://cite-seq.com/wp-content/uploads/2019/02/cite-seq_and_hashing_protocol_190213.pdf