ena-STUDY-IMPERIAL COLLEGE LONDON-14-08-2018-14:58:48:102-1203
FOXM1 ChIP Seq in HCT116 and DLD1 cells
HCT116 and DLD1 cells were grown to 90-95% confluency (150mm dish) and cross-linked with 1% formaldehyde for 10min at room temperature, 125mM of glycine was added for 5 min to stop cross-linking. The cells were washed with cold PBS and harvested using the cell lysis buffer with 1X PI (protease inhibitor). Following sonication, (10min for HCT116 and 12 min for DLD1) the samples were cleared by centrifugation (14,000g/10min/ 4°C), and supernatants were transferred into new tubes. Antibody binding (FOXM1 C-20 X, Santa Cruz biotechnology) was done using IgG protein-A and IgG protein-G dynabeads (Invitrogen) by incubating in a rotator for overnight in 4°C.  The dynabeads were then washed with TSE I + PI. DNA was eluted using elution buffer and decross linked the samples overnight at 650C or 6 hours. Extracted the DNA with Qiagen PCR purification kit and ran the samples in 7900 HT Fast Real-time PCR System (Applied Biosystems). The ChIP-ed DNA and input DNA were amplified as described above. Libraries were generated, and sequencing was performed on Illumina Hi Seq 2000 analyser according to the manufacturer's protocol. IGV (interactive genome viewer) was used to visualise ChIP sequenced data. The basic bed file format was used to visualise ChIP-seq peaks. The Cis-regulatory Annotation System (CEAS) function in Cistrome was used to functionally annotate binding sites.
HCT116 and DLD1 cells were grown to 90-95% confluency (150mm dish) and cross-linked with 1% formaldehyde for 10min at room temperature, 125mM of glycine was added for 5 min to stop cross-linking. The cells were washed with cold PBS and harvested using the cell lysis buffer with 1X PI (protease inhibitor). Following sonication, (10min for HCT116 and 12 min for DLD1) the samples were cleared by centrifugation (14,000g/10min/ 4°C), and supernatants were transferred into new tubes. Antibody binding (FOXM1 C-20 X, Santa Cruz biotechnology) was done using IgG protein-A and IgG protein-G dynabeads (Invitrogen) by incubating in a rotator for overnight in 4°C.  The dynabeads were then washed with TSE I + PI. DNA was eluted using elution buffer and decross linked the samples overnight at 650C or 6 hours. Extracted the DNA with Qiagen PCR purification kit and ran the samples in 7900 HT Fast Real-time PCR System (Applied Biosystems). The ChIP-ed DNA and input DNA were amplified as described above. Libraries were generated, and sequencing was performed on Illumina Hi Seq 2000 analyser according to the manufacturer's protocol. IGV (interactive genome viewer) was used to visualise ChIP sequenced data. The basic bed file format was used to visualise ChIP-seq peaks. The Cis-regulatory Annotation System (CEAS) function in Cistrome was used to functionally annotate binding sites.