3
nature research | reporting summary
April 2018Materials & experimental systems
n/a Involved in the study
Unique biological materials
Antibodies
Eukaryotic cell lines
Palaeontology
Animals and other organisms
Human research participantsMethods
n/a Involved in the study
ChIP-seq
Flow cytometry
MRI-based neuroimagingUnique biological materials
Policy information about availability of materials
Obtaining unique materials Samples no longer available, because they were consumed in the preparation for analysisAntibodies
Antibodies used EpCAM: Biolegend 9C4 antibody, Cat# 324208, Lot# B198775 , fluorochrome APC. CD45: Biolegend 2D1 antibody, Cat# 368516,
Lot# B264243, fluorochrome APC/Cy7. CD3: BD Biosciences SK7 antibody, Cat# 564001, Lot# 6109971, fluorochrome BUV395.Validation EpCAM (Biolegend 9C4 antibody): Product information: https://www.biolegend.com/en-us/search-results/apc-anti-human-
cd326-epcam-antibody-3758
CD45 (Biolegend 2D1 antibody): https://www.biolegend.com/en-us/search-results/apc-cy7-anti-human-cd45-antibody-12400
CD3 (BD Biosciences SK7 antibody): https://www.bdbiosciences.com/us/applications/research/t-cell-immunology/th-1-cells/
surface-markers/human/buv395-mouse-anti-human-cd3-sk7-also-known-as-leu-4/p/564001
Certificates of analysis for Biolegend lots are available at http://biolegend.com/it-it/certificate of analysis. A quality certificate
for the BD Biosciences lot is available at http://regdocs.bd.com/regdocs/searchCOAAction.do.Human research participants
Policy information about studies involving human research participants
Population characteristics The 14 patients studied by single-cell TCR and scRNA-seq are as follows: Lung1: age 65, female. Lung2: age 60, female. Lung3:^
age 51, female. Lung4: age 67, female. Lung5: age 71, female. Lung6: age 55, female. Endo1: age 59, female. Endo2: age 62,
female. Endo3: age 61, female. Colon1: age 47, female. Colon2: age 80, female. Renal1: age 65, male. Renal2: age 45, female.
Renal3: age 62, male. Population characteristics of clinical trial subjects are presented in their corresponding publications.Recruitment Patient samples were procured from third parties (Discovery Life Sciences, iSpecimen Inc., Avaden BioSciences, and TriMetis Life
Sciences) from adult patients undergoing resection surgery.Flow Cytometry
Plots
Confirm that:
The axis labels state the marker and fluorochrome used (e.g. CD4-FITC).The axis scales are clearly visible. Include numbers along axes only for bottom left plot of group (a 'group' is an analysis of identical markers).All plots are contour plots with outliers or pseudocolor plots.A numerical value for number of cells or percentage (with statistics) is provided.Methodology
Sample preparation Fresh surgical samples were separated into tumour and NAT compartments by the reviewing pathologist and shipped overnight
to our institution. Upon arrival, samples were rinsed with phosphate-buffered saline (PBS) until no traces of blood were visually
detected. Subsequently, samples were digested with a combination of Collagenase D (0.5mg/ml) and DNAse (0.1mg/ml) for
15min at 37°C with gentle shaking. Subsequently, samples were subjected to a gentleMACS dissociator (Miltenyi Biotec),
followed by an additional 10 min incubation at 37°C. Peripheral blood mononuclear cells (PBMCs) from patient blood samples
were isolated using 50mL Leucosep™ tubes (Greiner Bio-One International, Germany) and Ficoll-Paque™ PLUS (GE Healthcare,
Sweden). Whole blood drawn into sodium heparin blood collection tubes were diluted 3 times with PBS without calcium or
magnesium (Lonza, Walkersville, MD). Diluted cell suspensions were carefully layered on Leucosep tubes and centrifuged for 15
minutes at 800 x g at room temperature (RT). Interphase-containing PBMCs were harvested and washed with PBS and
subsequently centrifuged for 10 minutes at 250 x g at RT before further processing.Instrument Cells were purified by fluorescence-activated cell sorting (FACS) on a Becton Dickinson FACSAria Fusion cell sorter equipped with