Single cell RNAseq of HEC-50 Control (NT) and MAPK14-/- (MAP) spheroids.
MAPK14 +/+ and MAPK14 -/- HEC-50B cells were cultured as multicellular 3D spheroids for a duration of 12 days. At assay endpoint, 96 spheroids were pooled and dissociated with Accutase into a single-cell suspension. Samples were collected in biological triplicates.MAPK14 +/+ and MAPK14 -/- HEC-50B cells were cultured as multicellular 3D spheroids for a duration of 12 days. At assay endpoint, 96 spheroids were pooled and dissociated with Accutase into a single-cell suspension. Samples were collected in biological triplicates.Single-cell capture, library preparation and sequencing: Single cell suspensions were labelled with TotalSeq-B hashtag antibodies (BioLegend) following a modified version of the manufacturers sample multiplexing protocol. First, 1.5 uL of hashtag antibody was added to 150 uL of cells per sample and incubated on ice for 30 minutes. Cells were then pelleted and washed three times with 1.5 mL ice-cold 1X PBS (calcium and magnesium-free). Finally, cells were filtered through a 40 um cell strainer and stained with the viability markers Annexin V (BioLegend #640918) and SYTOX Blue (ThermoFisher #S34857) following manufacturers instructions. Single, live cells were then collected using a Sony SH800 cell sorter. Each hashtag-labeled, sorted sample was counted using the Luna-FL acridine orange/propidium iodide viability fluorescence assay (Logos Biosystems #F23001). Equal numbers of live cells were pooled from each sample, pelleted, and resuspended in 1X PBS (calcium and magnesium-free) + 0.04% BSA. The pooled sample was counted and then prepared for single cell capture, reverse transcription, and library preparation using the Chromium Next GEM Single Cell 3 v3.1 kit with Feature Barcoding for Cell Surface Protein according to the manufacturers user guide. The cell pool was loaded onto three lanes of the Chromium Next Gem Chip G targeting a total recovery of 30,000 cells (5,000 cells per sample). The libraries from each inlet were pooled and sequenced on NovaSeq 6000 S2 flow cell (Illumina) to a depth of 77,093 read pairs per cell. Each sample replicate can be identified using unique hashtag-labelled antibody sequence barcode. (NT R3-AGTAAGTTCAGCGTA  ; NT R4-AAGTATCGTTTCGCA ; NT R5- GGTTGCCAGATGTCA; MAP R3- GTCAACTCTTTAGCG ; MAP R4- TGATGGCCTATTGGG ; MAP R5-TTCCGCCTCTCTTTG  )