scRNA-seq

Cells were trypsinized and dissociated into a single-cell suspension and then kept on ice for all the downstream processing. After passing through a 40-μm cell constrainer and DAPI staining, live singlet cells were sorted into 96-well PCR plates with 4.2 μl of lysis buffer (mentioned above). At least three wells without cells were preserved as negative controls. The plate was quickly spun and further processed using the Smart-seq2 method²⁵.

To perform scRNA-seq on cells post-Live-seq extraction, cells were first cultured in a dish containing a silicone micro-insert (Ibidi, no. 80409) at a density of around 20 cells per well. The insert was then removed just before Live-seq sampling and all the cells were subjected to Live-seq extraction as described above, during a 1 h time window. To extract all the cells in this time, the extracted cytoplasm was not preserved. Then 1 and 4 h after the middle of the extraction time window (that is, 1 ± 0.5 and 4 ± 0.5 h postextraction), the cells, along with the control cells not extracted, were collected on ice and single cells were picked using a serial dilution approach. The downstream processing followed a similar workflow as the Smart-seq2 method.