To generate the Byrne et al data set, wild-type C57BL/6 mice were purchased from The Jackson Laboratory and housed at the University of Pennsylvania. Animal protocols were reviewed and approved by the Institute of Animal Care and Use Committee at the University of Pennsylvania. Mice were euthanized in a CO2 chamber using a flow meter to ensure CO2 was displaced at a rate of 30–70% of the chamber volume per minute and maintained for at least 1 minute after the loss of righting reflex is observed. Euthanasia was confirmed by bilateral thoracotomy. Animal handling information regarding tumor injections, drug prep and injection are included in Byrne et al (18 (link)).
To generate transgenic TCR data sets, spleen tissue from P14 and OT1 TCR transgenic mice (Nolz lab) were obtained. The spleen tissue was homogenized, treated with RBC lysis buffer and the resulting single-cell suspension was pelleted. Genomic DNA was extracted from the cell pellet using DNeasy blood and tissue kit (Qiagen).
To generate all the other data using animals, wild-type C57BL/6 mice were purchased from The Jackson Laboratory and maintained within the UCSF or OHSU laboratory for animal care barrier facility according to IACUC procedures. To generate the ST dilution series data set with mammary tumor and the Mesothelioma data set, we used mammary tumors from mouse mammary tumor virus (MMTV)-Polyomavirus middle T (PyMT) transgenic FVB/N mice and 40L orthotopic mesothelioma tumors respectively. Mammary tumors were resected from day 95 mice. For 40L orthotopic mesothelioma tumors, 2 x 106 cancer cells were injected i.p. into wild-type male C57BL/6 that were 6–12 weeks of age. For both tumor models, all mice were euthanized at a pre-defined end-stage for tissue harvest by cardiac puncture followed by cervical dislocation. These mice were cardiac perfused, under anesthesia using 1 –5% isoflurane, with 20 mL solution of heparin in PBS to clear tissues of residual blood followed by tissue harvest for further analysis including TCR sequencing. Tumor tissue was excised and flash frozen in liquid nitrogen and stored at −80 degree Celsius until further use for extracting genomic DNA for TCR sequencing. Murine SCC tumors were obtained from a previously published study, Medler et al (19 (link)).
To generate transgenic TCR data sets, spleen tissue from P14 and OT1 TCR transgenic mice (Nolz lab) were obtained. The spleen tissue was homogenized, treated with RBC lysis buffer and the resulting single-cell suspension was pelleted. Genomic DNA was extracted from the cell pellet using DNeasy blood and tissue kit (Qiagen).
To generate all the other data using animals, wild-type C57BL/6 mice were purchased from The Jackson Laboratory and maintained within the UCSF or OHSU laboratory for animal care barrier facility according to IACUC procedures. To generate the ST dilution series data set with mammary tumor and the Mesothelioma data set, we used mammary tumors from mouse mammary tumor virus (MMTV)-Polyomavirus middle T (PyMT) transgenic FVB/N mice and 40L orthotopic mesothelioma tumors respectively. Mammary tumors were resected from day 95 mice. For 40L orthotopic mesothelioma tumors, 2 x 106 cancer cells were injected i.p. into wild-type male C57BL/6 that were 6–12 weeks of age. For both tumor models, all mice were euthanized at a pre-defined end-stage for tissue harvest by cardiac puncture followed by cervical dislocation. These mice were cardiac perfused, under anesthesia using 1 –5% isoflurane, with 20 mL solution of heparin in PBS to clear tissues of residual blood followed by tissue harvest for further analysis including TCR sequencing. Tumor tissue was excised and flash frozen in liquid nitrogen and stored at −80 degree Celsius until further use for extracting genomic DNA for TCR sequencing. Murine SCC tumors were obtained from a previously published study, Medler et al (19 (link)).
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