Figure 4

DAM ChIP analysis of replicating and transcribing SV40 minichromosomes. 48 hour unfixed SV40 minichromosomes were analyzed by [a] standard chromatin immunoprecipitation using either IgG, or antibodies to H3K9me1, H3K9me2, H3K9me3, or Hyp H4 (7.5 μl each) bound to protein A agarose or [b] DAM ChIPs using antibody bound to magnetic beads in the first step which recognized RPA70 (SCBT; sc-25376) (50 μl) to immune select replicating minichromosomes or antibody which recognized RNAPII (SCBT; sc-900) (50 μl) to immune select transcribing minichromosomes. Following immune selection the magnetic beads were washed according to protocol and then divided into five equal aliqouts with an aliquot added to a tube which contained either IgG, or antibodies to H3K9me1, H3K9me2, H3K9me3, or Hyp H4 (7.5 μl each) bound to agarose. The immunoprecipitates bound to agarose from both analyses were then amplified by PCR (a: 32 cycles or b: 45 cycles) with primer sets to the early region of the SV40 genome as previously described [5–7]. Lane 1, ChIP with IgG; lane 2, ChIP with H3K9me1 (Abcam; ab9045); lane 3, ChIP with H3K9me2 (Abcam; ab1220); lane 4, ChIP with H3K9me3 (Abcam; ab8898); lane 5, ChIP with Hyp H4.