Reagent kit v3

Tumors harvested from mice were processed using the human tumor dissociation kit (Miltenyi Biotec, 130-095-929) with a GentleMacs Octo Dissociator with Heaters (Miltenyi Biotec, 130-096-427). Lungs from a single mouse were used for the control timepoint, lungs from two mice were pooled for the 2 week timepoint, lungs from two mice were pooled for the 4 week timepoint, lungs from one mouse were used for the 7 week timepoint, and the primary tumor from one mouse was used for the primary timepoint. OS-17 and niche cells expressing slp-mCherry were isolated using a BD Influx cell sorter using laser 610/20 BP. Single cell suspensions in 0.04% bovine serum albumin (BSA) (R&D Systems, S11150) -PBS of dissociated tumor tissues were generated and run on Chromium Single Cell 3′RNA-sequencing system (10X Genomics) with the Reagent Kit v3.1 (10X Genomics, PN-1,000,121) according to the manufacturer’s instructions. Briefly, cells were loaded into Chromium Next GEM Chip G Single Cell Kit (10X Genomics, PN-1,000,120) with a targeted cell recovery of 5,000–8,000 cells per sample. After performing cDNA purification, amplification, and library construction (sample index PCR cycles determined by protocol) as instructed, we sequenced sample libraries on a half lane of HS4000 (Illumina), SP, or NS_S2 to yield (after quality control) about 65,000 paired-end reads per cell.

Tumors and lungs harvested from mice were processed using the human tumor dissociation kit (Miltenyi Biotec, 130-095-929) with a GentleMacs Octo Dissociator with Heaters (Miltenyi Biotec, 130-096-427). Single-cell suspensions in 0.04% BSA-PBS of cell lines, dissociated tumor and lung tissues were generated and run on the Chromium Single Cell 3′RNA-sequencing system (10x Genomics) with the Reagent Kit v3.1 (10XGenomics, PN-1000121) according to the manufacturer’s instructions. Briefly, cells were loaded into Chromium Next GEM Chip G Single Cell Kit (10x Genomics, PN-1000120) with a targeted cell recovery of 5000 cells per sample. After performing cDNA purification, amplification, and library construction as instructed, we sequenced sample libraries on a half lane of HS4000 (Illumina) to yield (after quality control) about 65,000 paired-end reads per cell. For samples that contained lineage tracing barcodes 0.9 uL (100 µM) of Cellecta FSeqRNA-BC14 primer was added into the sample index pcr reaction.