Cell Sorting and RNA Isolation Protocol

The method was developed based on a previously published study (46 (link)). A step-by-step protocol for sample preparation, sorting, and RNA isolation is provided in Text S1. Key to successful RNA recovery is the gentle formaldehyde fixation at 4°C. Aliquots of fixed samples were adjusted to approximately 1.8 × 10⁷ cells/mL in 30 mL volume each and stained with SYBR green. Sorting of 5.4 × 10⁸ cells based on the FITC-signal (see above) directly into RNAprotect was performed with the BD FACSAria Fusion (BD Biosciences, Heidelberg, Germany). The sorted cells were collected on a filter from which RNA was extracted using a combination of Lysozyme and Proteinase K digestion with bead beating, and purified with NucleoZOL (TaKaRa Bio, Göteborg, Sweden). rRNA depletion was performed with the NEBNext Bacteria kit (NEB, Frankfurt, Germany). The libraries were prepared with the TruSeq kit (Illumina, San Diego USA).