Caenorhabditis elegans
Reconstitution of in vitro nucleosomes (invitrosomes): Naked genomic DNA from wild-type C. elegans (N2 strain) was isolated by digesting flash frozen worms with proteinase K (Roche, 2mg/ml final concentration) in worm lysis buffer (0.1M Tris-HCl at pH 8.5, 0.1 M NaCl, 50 mM EDTA, 1% SDS) at 65?°C for 45 min followed by phenol, phenol/chloroform, chloroform extraction and ethanol precipitation. RNA was removed by digesting the isolated nucleic acid with RNAse A (Roche) followed by phenol/chloroform, chloroform extraction and ethanol precipitation. To produce DNA templates for both the Rsa I and Hinc II experiments, 40 µg of high-molecular weight genomic DNA was digested with 200 units of either restriction enzyme Rsa I or Hinc II (New England BioLabs) with the supplied buffers and 1X BSA (New England BioLabs). Digestion proceeded at 37?°C for two hours followed by phenol, phenol/chloroform, chloroform extraction and ethanol precipitation, resulting in complete digestion as assayed on a 1% UltraPure Agarose (Invitrogen) gel. A continuous smear of fragments was seen for both digestions with a distribution of fragments lengths centered upon and enriched around 850?bp and 3500 bp for the Rsa I and Hinc II digestions respectively.   The Rsa I and Hinc II DNA digestions were assembled with recombinant Xenopus histones (a gift from Geeta Narlikar) into nucleosomes as described previously (Zawadzki KA, Morozov AV, Broach JR: Chromatin-dependent transcription factor accessibility rather than nucleosome remodeling predominates during global transcriptional restructuring in Saccharomyces cerevisiae. Molecular biology of the cell 2009, 20:3503-3513) at a 1.1:1 molar ratio of DNA to histone octamer, such that on average one nucleosome would form per 850?bp or 3500 bp of DNA for the Rsa I and Hinc II reconstitutions, respectively. Specifically, 9.13?µg of DNA and 1.45?µg of histone octamer (for Rsa I) and 22.00?µg of DNA and 0.88?µg of histone octamer (for Hinc II) were reconstituted in a total volume of 200?µl. We call these in vitro reconstituted nucleosomes invitrosomes and will refer to them as such hereafter.       Isolation of invitrosome core DNA fragments: Invitrosome core DNAs from both Rsa I and Hinc II reconstitutions were isolated by diluting 60?µl of the respective invitrosomes into a total volume of 200?µl containing 5?mM MgCl2, 5?mM CaCl2, 70?mM KCl and 10?mM Hepes at pH?7.9 (final concentrations) and digesting with 20 units of micrococcal nuclease (Roche) resuspended at 1?U/µl for 15?min at room temperature. The digestion was stopped by adding an equal volume of 3% SDS, 100?mM EDTA and 50?mM Tris. Histones were removed by treating with one-tenth volume proteinase K (20?mg/ml in TE at pH?7.4) for 30?min at 50?°C followed by phenol/chloroform and chloroform extractions and ethanol precipitation. In order to obtain enough sample for the Hinc II reconstitution, this procedure was repeated three times to process the entire Hinc II invitrosome sample and then pooled together in a total volume of 30 µl (the single processing of the Rsa I sample was also in a total volume of 30 µl). Invitrosome DNA cores were assayed for complete digestion and isolated on a 2% UltraPure Agarose (Invitrogen) gel run at 100?V for 1?h followed by DNA extraction from the gel using a QIAquick Gel Extraction Kit (Qiagen) following the standard protocol, with the exception of allowing the isolated gel sample to incubate in Buffer QG at room temperature until dissolved, rather than heating the sample at 50?°C as recommended by the manufacturer.   Illumina library preparation and sequencing. The Rsa I and Hinc II libraries were prepared by processing the invitrosome core DNA fragments using the Illumina Genomic DNA Sample Prep Kit (Illumina 2007 Rev. A). Fragment end repair, linkering and library amplification were all done according to the kit instructions “Preparing Samples for Sequencing Genomic DNA” (Illumina 2007 Rev. A), with exceptions to the protocol mentioned below. Since our libraries are composed of ~147 bp DNA cores rather than intact genomic DNA, the protocol was started at the “Perform End Repair” step (page 11). At this step, 20 µl of the Rsa I invitrosome core DNA sample and 30 µl of the Hinc II invitrosome core DNA sample were used. For the Rsa I sample, 10 µl of extra water was added to achieve the 30 µl volume prescribed by the protocol. At the “Ligate Adapters to DNA Fragments” step the purification was performed with the QIAquick PCR Purification Kit (Qiagen) rather than MinElute PCR Purification Kit, and eluted in 30 µl of EB rather than 10 µl, and 10 µl of the sample was used in the next step.  Additionally, a no-DNA control sample was processed in parallel to the Rsa I and Hinc II samples. After library preparation, each library was sequenced using a single lane of the Illumina GAII sequencer, resulting in 9.5 million and 5.5 million raw 36-bp reads for the Rsa I and Hinc II libraries, respectively. 