Single-Cell RNA-Sequencing of Live Cells

Cells were resuspended in RPMI media to obtain a single-cell suspension with high cell viability. Next, cells were stained with a live/death dye (DAPI) and dead cells were removed using fluorescence-activated cell sorting (FACS). Live cells were resuspended in PBS buffer and recounted using AOPI staining and the Nexcelom Cellometer Auto 2000 Cell Viability Counter. Finally, cells from four independent biological replicates were pooled in equal cell numbers into a single-cell suspension for each condition. Cell suspensions were processed for single-cell RNA-sequencing using the 10×-Genomics 3′ v2 kit^{33 (link)}, as specified by the manufacturer’s instructions. Namely, 1 × 10⁴ cells from each condition were loaded in separate inlets of a 10×-Genomics Chromium controller in order to create GEM emulsions. The targeted recovery was 3000 cells per condition. Emulsions were used to perform reverse transcription, cDNA amplification and RNA-sequencing library preparation. Libraries were sequenced on the Illumina HiSeq 4000 platform, using 75 bp paired-end reads and loading one sample per sequencing lane.