Oxidative stress is recognazed as a hallmark of cancer. However, little is known about intratumor heterogeneity of reactive oxygen species (ROS) and its influence on tumor behavior, largely due to technical challenges inherent in detecting intratumor ROS at single-cell resolution. We have developed a tumor-targeted H2O2 probe (T-AP1) by conjugating trastuzumab with both Alexa Fluor 647 (AF647), an H2O2-insensitive fluorophore, and peroxy green 1 (PG1), an irreversible H2O2-sensitive chemical probe to provide a “ratiometric” readout, allowing H2O2 measurements independent of accumulated T-AP1 levels. Given the ability of T-AP1 to persistently mark tumor cells that experience H2O2 exposure, we employed cell sorting to isolate tumor cells with high (PG1highAF647+) and low (PG1lowAF647+) H2O2 exposure levels from T-AP1–labeled NCI-N87 xenograft tumors. PG1highAF647+ and PG1lowAF647+ cells obtained via cell sorting from three biologically independent N87 tumors were subjected to RNA sequencing. Overall design: N87 xenograft tumors were resected from male athymic nude mice (BALB/c Slc-nu/nu) 3 weeks post-subcutaneous inoculation. T-AP1 was administered 12 hours before the harvestion of the tumors. Resected tumors were minced into small fragments in ice-cold RPMI 1640 containing DNase I, Liberase DH (Roche), and catalase. The fragments were incubated in a shaker for 1 h at 37°C and filtered through 40 μm cell strainers (BD Falcon). Filtrated cells were suspended in PBS and stored on ice until cell sorting. The cells were treated with 5 μM propidium iodide (PI, Dojindo) for 10 min to exclude dead cells, and PI-negative cells were sorted according to the fluorescence intensity of PG1 and AF647. Total RNA from the sorted tumor cells was purified using an RNeasy Micro kit (Qiagen). cDNA libraries were prepared using a SMART-Seq v4 Ultra Low Input RNA Kit for sequencing (Takara Bio). Sequencing libraries were then prepared with Nextera XT Index Kit v2 SetA/B/C/D (Illumina). The prepared library was sequenced on NovaSeq 6000 system (Illumina).