Illumina HiSeq 2500 sequencing; Chip-Seq of wild-type GATA3 and two mutants (335fs, 308*) in the ZR75-1 human breast cancer cell line
Chip-Seq of wild-type GATA3 and two mutants (335fs, 308*) in the ZR75-1 human breast cancer cell line
Approx 8 million adherent cells were crosslinked for 10 mins in 1% formaldehyde. Cross linking was stopped by adding glycine to 0.125M. Cells were lysed in Farnham Lysis Buffer, washed in PBS and nuclei were lysed with RIPA buffer. ZR75-1 cells were cultured in RPMI 1640 supplemented with 10% serum Cell lines were generated by retroviral transduction of ZR75-1 with vectors containing either GATA3 WT, 308 or 335 mutations.  Samples were sonicated using a Diagenode XL Bioruptor to fragments of 200-400 bp. Chromatin immunoprecipitation was performed using anti-FLAG antibody bound to protein G magnetic dynabeads. DNA fragments recovered from chromatin immunoprecipitation were reapired using Klenow DNA polymerase, liganted to Illumina TruSeq adapters using T4 DNA ligase and size selected for DNA with adapters attached.