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Single cell clones were grown for immunoblot analysis and those showing loss of ATF4 protein were selected for sequence analysis involving the isolation of genomic DNA (Qiagen, 69504), PCR amplification using KOD Xtreme™ Hot Start DNA Polymerase (Millipore, 71975) and primers TCGATGCTCTGTTTCGAATG and CTTCTTCCCCCTTGCCTTAC flanking the targeted deletion site, and sequencing on an ABI3730xl DNA analyzer at the DNA Resource Core of Dana-Farber/Harvard Cancer Center (funded in part by NCI Cancer Center support grant 2P30CA006516-48)
