RNA sequencing sample preparation

Single cell suspensions of metastatic lungs from control or irradiated mice (n=10 per group, pooled) were prepared as described above. CD45^- Ter119^- cells were sorted by flow cytometry following staining with anti-mouse CD45, Ter119 and DAPI. Library generation for 10x Genomics analysis were performed using the Chromium Single Cell 3’ Reagents Kits (10x Genomics), followed by sequencing on an Hiseq4000 (Illumina) to achieve an average of 50,000 reads per cell.

Lung CD45^-CD31^-Ter119^-EpCAM⁺ and CD45^-CD31^-Ter119^- EpCAM^- cells were sorted from control, irradiated or neutrophil-depletion irradiated mice 7 days post-irradiation by flow cytometry. Total RNA was isolated using the miRNeasy Micro Kit (Qiagen, cat# 217084), according to the manufacturer’s instructions. Library generation was performed using the KAPA RNA HyperPrep with RiboErase (Roche), followed by sequencing on a HiSeq (Ilumina), to achieve an average of 30 million reads per sample.