Flura-Seq Identifies In Situ Transcriptomes of Micrometastases

Understanding the biology of rare cell populations in the context of their microenvironment requires an accurate analysis of the transcriptomes of these cells as expressed in situ. We developed fluorouracil-tagged RNA sequencing (Flura-seq) to characterize the transcriptomes of small cell subpopulations from a whole organ in model systems. The method utilizes cytosine deaminase (CD), which converts the non-natural pyrimidine base fluorocytosine to fluorouracil. Expression of S. cerevisiae CD in cells of interest and exposure to fluorocytosine generates fluorouracil, which is metabolically incorporated into newly synthesized RNAs. The fluorouracil-tagged RNAs can then be immunopurified and sequenced. We applied Flura-seq to define the transcriptome of human breast cancer xenografts, representing as few as 0.003% of host organ cell population during the early stages of metastatic colonization of mouse lungs. The robustness, simplicity and lack of toxicity of Flura-seq make this tool broadly applicable to many studies in developmental, regenerative, and cancer biology. Overall design: RNAs were tagged withfluorouracil, and the tagged RNAs were immunopurified before sequencing. MDA_Con-Dox = MDA231 control cells that does not express CD/UPRT MDA_FC50uM4hr = MDA231 cells treated with Doxycycline for 24 hours to express CD/UPRT and further treated with 50 uM 5-fluorocytosine for 4 hours. MDA_FC50uM12hr = MDA231 cells treated with Doxycycline for 24 hours to express CD/UPRT and further treated with 50 uM 5-fluorocytosine for 12 hours. MDA_FC250uM4hr = MDA231 cells treated with Doxycycline for 24 hours to express CD/UPRT and further treated with 250 uM 5-fluorocytosine for 4 hours. MDA_FC250uM4hr = MDA231 cells treated with Doxycycline for 24 hours to express CD/UPRT and further treated with 250 uM 5-fluorocytosine for 12 hours. MDA_SBCon = MDA231 cells treated with 2.5 uM SB-505124 for 150minutes. MDA_TGFBCon = MDA231 cells treated with 200 pM TGFB for 150minutes. MDA_SB_IP = MDA231 cells treated with Doxycyline for 24 hours to express CD/UPRT, then with 250 uM of 5-fluorocytosine for 30 minutes before adding 2.5 uM of SB-505124 for additional 150 minutes, and the mRNAs from the cells were immunoprecipitated with anti-BrdU antibody. MDA_TGFB_IP = MDA231 cells treated with Doxycyline for 24 hours to express CD/UPRT, then with 250 uM of 5-fluorocytosine for 30 minutes before adding 200 pM of TGFB for additional 150 minutes, and the mRNAs from the cells were immunoprecipitated with anti-BrdU antibody. MDA_msLung_4h Input = MDA231 cells injected in mouse lung through tail vien, and 4 weeks after the injection CD/UPRT was induced by feeding Doxycycline diet for 2-3 days, and 250 mg/kg 5-fluorocytosine was injected intraperitoneally and 125 mg/kg thymine subcutaneously, and 4 hours later mouse lungs were harvested, homogenized, mRNAs were isolated and sequenced. MDA_msLung_4h IP = MDA231 cells injected in mouse lung through tail vien, and 4 weeks after the injection CD/UPRT was induced by feeding Doxycycline diet for 2-3 days, and 250 mg/kg 5-fluorocytosine was injected intraperitoneally and 125 mg/kg thymine subcutaneously, and 4 hours later mouse lungs were harvested, homogenized, mRNAs were isolated, immunopurified with anti-BrdU antibody and sequenced. MDA_msLung_12h Input = MDA231 cells injected in mouse lung through tail vien, and 4 weeks after the injection CD/UPRT was induced by feeding Doxycycline diet for 2-3 days, and 250 mg/kg 5-fluorocytosine was injected intraperitoneally and 125 mg/kg thymine subcutaneously, and 12 hours later mouse lungs were harvested, homogenized, mRNAs were isolated and sequenced. MDA_msLung_12h IP = MDA231 cells injected in mouse lung through tail vien, and 4 weeks after the injection CD/UPRT was induced by feeding Doxycycline diet for 2-3 days, and 250 mg/kg 5-fluorocytosine was injected intraperitoneally and 125 mg/kg thymine subcutaneously, and 12 hours later mouse lungs were harvested, homogenized, mRNAs were isolated, immunopurified with anti-BrdU antibody and sequenced.