BMC Genomics 2007, 8:139 http://www.biomedcentral.com/1471-2164/8/139 in transduction of naïve PC12 cells plated on collagen. Selection using 6 µg/mL blasticidin was initiated and maintained for 7 days until stable lines were enriched. Cells were maintained for 14 days before differentiation and experimentation. Experiments using stable shRNA- expressing PC12 cells were compared to wild-type PC12 cells of the same original population and passage number in all cases. Western blotting Sample preparation for SDS-PAGE was performed as pre- viously described [87]. Antibodies used were against nes- tin (Rat401, Pharmingen), dopamine transporter (D2442, Sigma), p53 (pAb122, Pharmingen), phospho- serine 15 p53 (No. 9284, Cell Signaling, Davers, MA) and actin (MAb1501, Chemicon). Proteins were visualized using either ECL reagent (GEH Amersham, Piscataway, NJ) or SuperSignal reagent (Pierce, Rockford, IL). Chromatin immunoprecipitation assays ChIP assays were performed similarly to previous reports [88,89]. Briefly, NGF-differentiated and naïve (dividing) PC12 cells were crosslinked by 1% formaldehyde with rocking for 15 minutes before terminating the crosslink- ing reaction with 125 mM glycine. Cells were washed, lysed by Dounce homogenization and resupended in a buffer containing sodium deoxycholate, SDS and Triton X-100 [88]. Chromatin was sheared to an average length of 500 bp by sonication on ice and samples were clarified by centrifugation at 10,000 × g. Sheared, crosslinked chro- matin complexes were precleared with protein A agarose. p53-DNA complexes were immunoprecipitated using anti-full length p53 polyclonal antibody (FL-393, Santa Cruz Biotech, Santa Cruz, CA) or with control rabbit IgG (I-5006, Sigma). Immune complexes were captured with protein A agarose and beads were washed as described [88,89]. Protein/DNA complexes were eluted and crosslinks reversed by incubating at 65°C for at least 6 hours. To assay for p53 binding sites in purified ChIP DNA, target specific primers [See Additional file 1; Table 2] were used to measure amounts of target sequence in immunoprecipitated samples by qPCR using SYBR Green- based detection (BioRad, Richmond, CA). Experimental qPCR values were normalized against values obtained for 25 ng of input DNA using the same primer set. Non-spe- cific enrichment of chromatin was minimal as determined by the amplification of enriched ChIP DNA using primers designed to multiple non-transcribed genomic regions. Cloning and analysis of enriched ChIP DNA Cloning of enriched ChIP DNA was performed by using Genpathway's (San Diego, CA) FactorPath Discovery approach, as previously described [89]. Enriched ChIP DNA was incubated with T4 DNA polymerase to generate blunt ends, ligated to a DNA adapter X consisting of annealed 25-nt and 11-nt oligonucleotides [90], and PCR amplified using the 25-nt adapter X oligonucleotide as primer. A 40–50 bp tag library was generated from the amplified ChIP DNA by the strategy described in [90]. Amplified ChIP DNA was mixed with "random primer A" consisting of a fixed sequence A (containing a Bgl II restriction site) and a randomized 9-nt sequence at the 3' end. After denaturation of the DNA followed by chilling of the sample on ice, the annealed random primers were extended using Klenow DNA polymerase [90]. Extension products were purified on Sephacryl S-400 HR spin col- umns (GE Healthcare, Chalfont St. Giles, UK) and mixed with "random primer B", which differed from "random primer A" by having a different fixed sequence B (also containing a Bgl II restriction site). For second strand syn- thesis, denaturation, incubation with Klenow DNA polymerase and purification on Sephacryl S-400 HR was repeated as described above for first strand synthesis. The resulting DNA was amplified by PCR using primers A and B, and amplified DNA was electrophoresed on a native polyacrylamide gel (BioRad). DNA fragments of 80–90 bp were isolated, re-amplified with primers A and B and sub- jected to a second round of gel-purification. The DNA was then digested with restriction endonuclease Bgl II to excise the 40–50 bp tags representing genomic sequences. After gel-purification, tags were concatemerized by ligation in the presence of a limiting amount of adapter C containing a Bgl II-compatible 4-nt overhang on one end and a blunt- end on the other end. Ligation products were amplified using the adapter C sequence as PCR primers, and prod- ucts containing 10–15 tags were gel-purified, cloned into the pCR2.1-TOPO vector (Invitrogen) and sequenced. A detailed diagram [see Additional file 1; Figure 5], summa- rizes these procedures for cloning and analysis of enriched ChIP DNA. Individual tag sequences were aligned to NCBI Rat Genome Build v3.4 using MEGABLAST v2.2.11. Tags producing low-scoring or multiple alignments were eliminated from consideration. Clusters were generated by grouping of tags mapping to within 2 kb of each other. Alignments were associated with annotated or predicted genes mapping to within 10 kb of each tag. The presence of a consensus p53 binding site was determined in each tag by querying 500 bp of bilateral genomic sequence flanking each tag using the p53MH algorithm [47]. Func- tional assessment of genetic elements associated with individual tags was performed using GoMiner [91] and grouped into ontological subsets for further analysis and selection. To confirm candidate p53 binding sites, PCR primers targeting a region within 200 bp of each selected alignment or cluster [See Additional file 1; Table 2] were used to measure the amount of sequence in immunopre- cipitated sample by qPCR as described above. Page 14 of 17 (page number not for citation purposes)