In vitro growth signature identification from SmartSeq3 sequencing of 5 human cancer cell lines and 1 mouse cancer cell line

Cell lines used to determine the growth of cell lines over time were used for transcriptomic profiling to build a model that predicts growth rate from transcriptome of cells. To establish robust transcriptional signatures of dying cells, we performed single-well cell sorting followed by Smart-seq3xpress. 4T1, A549, MDA-MB-231 clone A, L and S were grown in cell culture medium. Adherent MDA-MB-231 clone L were grown with 1uM of Paclitaxel, a concentration we have previously shown to slow proliferation while retaining a viability compatible with the experiment27. The cells in culture were detached with Trypsin treatment for 3-5 minutes at 37 degrees C and the reaction was quenched by adding back the cells supernatant to conserve dead cells. The suspension was washed twice in PBS, 4 minutes at 400g. The cells were then labelled using the live/dead stain BOBO3 and apoptosis marker AnnexinV-APC following manufacturer's instruction. We isolated 3 cell populations on the basis of the BOBO3 and Annexin-V flow cytometry markers using an Aria3 flow cytometry with a 100um nozzle and low pressure settings. Live cells were gated as BOBO3- AnnexinV-, dying (early apoptotic) cells as BOBO3- AnexinV+ and dead cells (necrosis or late apoptosis) as BOBO3+ AnexinV+ (Fig. S2a-b). Individual cells were isolated in 384 well pcr-plates. After 10s centrifugation at 1000g the plates were sealed and stored at -80*C until processed using Smart-seq3xpress protocol30. All the reagents and bioinformatic pipeline related to Smart-seq3xpress are available online31.