HeLa cells were cultured under normal growth conditions (10% Fetal Bovine Serum).
Biomaterial provider
Iyer lab, UT-Austin
Extracted molecule
genomic DNA
Extraction protocol
STAGE is based on high-throughput sequencing of concatemerized tags of defined length that are derived from DNA enriched by ChIP. Proteins were cross-linked to their binding sites in vivo with formaldehyde and chromatin was extracted and sheared. The cross-linked protein-DNA complexes were immunoprecipitated, cross-links were reversed and ChIP DNA was amplified by PCR using biotinylated primers. Amplified DNA fragments were digested with NlaIII, which cuts at 5'-CATG sites. Fragments with ends containing the NlaIII site were isolated by binding to streptavidin beads. They were separately ligated to one of two linkers containing a MmeI site, then incubated with MmeI, which cleaves 21 bp away from its recognition site. The 21-bp tags attached to linkers were isolated and ligated to create ditags. Ditags were amplified by PCR using nested primers and trimmed by digesting with NlaIII. Trimmed ditags were gel purified, concatemerized by ligation, cloned and sequenced
Description
ChIP procedure Human cells were cross-linked by addition of formaldehyde (1% final concentration) directly to tissue culture plates for 7 min at room temperature. Cross-linking was terminated by adding glycine to a final concentration of 125 mM. Cells were washed with cold phosphate-buffered saline (PBS) containing PMSF, scraped off the plates, collected by centrifugation and washed again. After centrifugation, the pellet was resuspended in SDS lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-Cl pH 8.1, plus protease inhibitors) and incubated at room temperature for 20 min. Cells were sonicated on ice and fragmented DNA was visualized on an agarose gel (average size 1 kb). The sample was centrifuged at 12000 rpm at 4 °C for 10 min and 10x ChIP dilution buffer (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-Cl pH 8.1, 150 mM NaCl, plus protease inhibitors) was added to the collected supernatant. Sample was pre-cleared with protein A-agarose beads (previously washed with 10x ChIP dilution buffer) at 4 °C for 1 hr. Pre- cleared chromatin was incubated with an antibody against E2F4 (sc-1082x, Santa Cruz) at 4 °C overnight. For the mock IP controls in Figs. 3 and 4, the antibody was left out. Pre-washed protein A-agarose beads were added and protein-DNA complexes were recovered after a 2 hour incubation at 4 °C. Immunoprecipitated complexes were successively washed with Low- salt wash buffer (0.1% Deoxycholate, 1% Triton X-100, 1 mM EDTA, 50 mM HEPES pH 7.5, 150 mM NaCl), High-salt wash buffer (0.1% Deoxycholate, 1% Triton X-100, 1 mM EDTA, 50 mM HEPES pH 7.5, 500 mM NaCl), LiCl wash buffer (250 mM LiCl, 0.5% NP-40, 0.5% Deoxycholate, 1 mM EDTA, 10 mM Tris-Cl pH 8.1) and TE buffer (10 mM Tris-Cl pH 7.5, 1 mM EDTA). SDS elution buffer was added and incubated at 65 °C for 30 min to recover protein-DNA complexes. Crosslinks were reversed by incubating at 65 °C overnight. The sample was treated with RNase A and Proteinase K, extracted with phenol:chloroform and precipitated. The pellet was resuspended in 25 µl of water. A 5'-biotinylated primer was used for random amplification of immunoprecipitated DNA fragments. The biotinylated amplified ChIP material was used as input for STAGE.
Genome wide mapping of transcription factor downstream targets using STAGE
Data table header descriptions
TAG
Each tag is a 17 bp signature sequence derived from ChIP enriched DNA. Immunoprecipitated DNA was digested with NlaIII, ligated with linkers containing the MmeI restriction site and digested with MmeI to release 21 bp tags. Tags were then concatamerized, cloned and sequenced to generate the STAGE sequencing pool for the given transcription factor.
COUNT
This is the frequency of the tag in the STAGE sequencing pool.
