cohesin depletion which suggest that cohesin is a stabilising factor in chromatin looping. It will therefore be important to test if these mutations affect higher order chromatin structure at specific loci. Although such defects may be very subtle, they could at some loci cause defects in gene regulation during development. Another important goal for the future will be to determine at the genomic level which CTCF-cohesin sites can interact with each other, if these interactions change during cell differentiation and how such changes might be specified. Methods IGF2-H19 methylation and expression analysis Methylation analysis was by bisulphite- and pyrosequencing with primers as described previously [23,25,51]. Expression analysis was done by qPCR on reverse transcribed RNA. Primers for qPCR were IGF2 Fwd CTCACCTTCTTGGCCTTCG, IGF2 Rev GGAAACAGCACTCCTCAACG, H19 t Fwd GAGATT- CAAAGCCTCCACGACT and H19 Rev GCGTAATG- GAATGCTTGAAGG. B Actin was analysed using primers from a QuantiTect Assay (Qiagen). Quantitation was done by extrapolation to standard curves for the pimers. Wilcoxon signed rank tests were done to compare paired RNAi and control samples when n$3. P#0.05 was considered significant. ChIP Chromatin Immunoprecipitation (ChIP) was done as described previously [6]. Input and immunoprecipitated (IP) material was quantified by Picogreen (Invitrogen), and real-time PCR with standard curves was performed. Values were corrected for DNA amount, and enrichment was calculated as IP over input. When comparing ChIP from cohesin depleted cells with control cells (Figure 6C), the IP/Input was further normalised against a region where CTCF does not bind (IGF2 exon 9). ChIP primers are shown in Table S2. 3C Quantitative 3C was described previously [31,36,52,53] and performed with the following modifications: 56106 of cells were cross-linked in 1% formaldehyde at 37uC for 10 minutes. After washing in PBS, the cells were lysed on ice in lysis buffer (Tris-HCl (pH = 8) 50 mM, SDS 1%, EDTA 10 mM) for 10 minutes. Nuclei were recovered by centrifugation and resuspended in BamHI digestion buffer (New England Biolabs (NEB)), supplemented with Triton-X100 to a final concentration of 1.8% and incubated 1 h at 37uC. 1.56106 nuclei were digested overnight with 1000 U of BamHI (NEB) in a 300 ml reaction volume. Digestion efficiency at each BamHI restriction site within the locus was assessed by qPCR across each restriction site. The percentage of digestion was determined by comparing template amplification of digested and undigested fractions (not religated) after normalising to copy number as previously described [31]. All regions within the locus were digested equally efficiently. This step was an important quality control check, and if digestion was below 70% the chromatin was discarded. Ligation was carried out on 2.5 ng/ml digested chromatin in a 1.5 ml reaction volume of T4 ligase buffer containing 3200 U of T4 ligase (NEB). A further overnight digestion step with 1000 U of EcoRI (which cuts outside the hybrid religated products) was incorporated prior to reversal of cross links, phenol chloroform purification and ethanol precipitation. This step is necessary to remove possible qPCR bias caused by size differences in the religated products. Cohesin Mediates Chromatin Conformation 3C PCR primers flanking restriction sites were designed to have similar melting temperatures, and the PCR efficiency of each primer combination was assessed on a PCR standard template. A stock of PCR standard template was prepared similar to that described previously [52] by amplification of 36 genomic regions across the IGF2-H19 locus on commercially obtained genomic DNA (Becton Dickinson (BD)). These amplicons were column purified and quantified using Nanodrop UV spectroscopy. Equimolar amounts of amplicons were mixed, BamHI digested, re-ligated, phenol-chloroform extracted, ethanol precipitated, dissolved in H2O and stored at 220uC. Q-PCR was done with Sybr-green (ABI Power SYBR) on a 384 well real time machine (7900HT Fast Real time PCR system, Applied Biosystems). Quantitative determination of association frequencies was essen- tially done as described [31]. Copy number of 3C template was determined by qPCR amplification of a region between IGF2 and H19 which did not have BamHI restriction sites (Chr11: 2057922– 2057991, Ensembl). Template copy number was used to ensure that the amount of 3C template was within the range of the standard curve for any given product. All interaction frequencies were normalised to the circularisa- tion frequency of the i-fragment as internal digestion-ligation control. We verified our normalisation method by including a b- actin gene region (Chr7: 5,326,283–5,357,206) that contained 3 BamHI restriction sites and compared the outcome of the 3C association frequencies across the locus when normalised to adjacent BamHI sites (2–3 B-actin), alternative BamHI sites (1–3 B- actin), or alternative internal sites within the IGF2-H19 locus (data not shown). 3C primers and combinations are in Tables S2 and S4. Biological replicates for siRNA 3C experiments were done by splitting test and control cells each into 3 equal aliquots prior to synchronisation. After harvesting the cells and digesting the chromatin for 3C experiments, the replicate templates were evaluated for digestion efficiency and normalised for equal amounts before ligation. Prior to PCR amplification, the replicate templates’ copy numbers were determined by qPCR as described above, and equal amounts (copy number) of DNA recovered after 3C for control and RNAi template was used for the 3C qPCR. Association frequencies were normalised to the circularisation frequency of the i-fragment as described above. Normalisation data for 3 biological replicates are shown in Figure S5. The frequency of circularisation of the i-fragment is similar in controls and RNAi treated cells (Figure S5A), confirming that RNAi treatment for cohesin depletion did not affect digestion or ligation efficiency. Using the circularisation frequency of the i-fragment to normalise an association frequency between the enhancer anchor (primer m) and a restriction site located between the enhancer and CTCF DS (restriction site p), shows no significant differences between RNAi treated cells and controls (Figure S5B). This is as expected, because there is no binding of CTCF or cohesin to the enhancer and its interaction with restriction site p is due to random ligation. Circularisation of the i-fragment was therefore a suitable internal normaliser. ChIP loop ChIP loop was performed as follows: 3 aliquotes of 56106 cells were first fixed in formaldehyde and nuclei were prepared as for 3C. One aliquot was used for 3C and the remaining aliquots were briefly sonicated to produce chromatin fragments of 500 bp and then digested overnight with BamH1 as for 3C protocol. After digestion the nuclei were pre-cleared on agarose beads (UP- STATE) and immunoprecipitated with antibodies to cohesin (anti- SMC3 [6,54] and CTCF (UPSTATE) as described in the ChIP PLoS Genetics | www.plosgenetics.org 12 November 2009 | Volume 5 | Issue 11 | e1000739