ena-STUDY-ROSWELL PARK CANCER INSTITUTE-31-03-2017-04:22:54:119-142
Effect on gene expression after 4 days of a single, 15 Gy dose of ionizing X ray radiation on H522 human lung cancer, and FaDu, HSC2 and SCC4 human head and neck cancer cell-lines
H522 human lung cancer, and FaDu, HSC2 and SCC4 human head and neck cancer cells were grown adherent to 60%-80% confluence on 10-cm dishes in 8 ml RPMI-1640/+DMEM medium with 10% fetal bovine serum. Cells were irradiated with one dose of 15 Gy ionizing X rays in a Faxitron FX-650 machine; control cell culture dishes were not irradiated. After a day of radiation, adherent cells were detached by trypsinization and replated on 10-cm dishes in 8 ml RPMI-1640/+DMEM medium with 10% fetal bovine serum to reach 70%-90% confluence after three days, when adherent cells were harvested by scraping. Harvested cells were pelleted and kept frozen at -80 ºC. For each cell-line, three experiments were performed to obtain three pairs of control and irradiated cell pellets. One µg of total RNA isolated from each of the cell pellets using Norgen-Biotek Total RNA Isolation kit with an on-column DNAse I treatment step was used to prepare sequencing library for directional RNA sequencing to obtain paired 76 b reads on an Illumina NextSeq 500 instrument. Twenty-four libraries were sequenced in each flow lane, and each library was sequenced in four lanes of one flow cell. On-system software was used to demultiplex sequencing data.
H522 human lung cancer, and FaDu, HSC2 and SCC4 human head and neck cancer cells were grown adherent to 60%-80% confluence on 10-cm dishes in 8 ml RPMI-1640/+DMEM medium with 10% fetal bovine serum. Cells were irradiated with one dose of 15 Gy ionizing X rays in a Faxitron FX-650 machine; control cell culture dishes were not irradiated. After a day of radiation, adherent cells were detached by trypsinization and replated on 10-cm dishes in 8 ml RPMI-1640/+DMEM medium with 10% fetal bovine serum to reach 70%-90% confluence after three days, when adherent cells were harvested by scraping. Harvested cells were pelleted and kept frozen at -80 ºC. For each cell-line, three experiments were performed to obtain three pairs of control and irradiated cell pellets. One µg of total RNA isolated from each of the cell pellets using Norgen-Biotek Total RNA Isolation kit with an on-column DNAse I treatment step was used to prepare sequencing library for directional RNA sequencing to obtain paired 76 b reads on an Illumina NextSeq 500 instrument. Twenty-four libraries were sequenced in each flow lane, and each library was sequenced in four lanes of one flow cell. On-system software was used to demultiplex sequencing data.