GSM1238658: WT culture, anti-RpaA, ZT 28 h, replicate 1; Synechococcus elongatus PCC 7942; ChIP-Seq
Samples were thawed on ice and resuspended in 500-600 ul of ice-cold lysis buffer (50 mM HEPES pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate, and 1x Roche Complete EDTA-free Protease Inhibitor Cocktail). Cells were lysed by beadbeating at 4 °C in 2 ml screw-top tubes with 0.1 mm glass beads for 10 cycles of 30 seconds each separated by at least 30 sec of cooling on ice. Lysate was separated from the beads by piercing the bottom of each tube with a small-diameter needle, placing the tube into a clean 1.5 ml microcentrifuge tube, and centrifuging for several minutes at < 500 x g at 4 °C to transfer the lysate to the 1.5-ml tube. Chromatin was then sheared to a length of ~300 bp by sonication on ice in a Misonix sonicator 3000 for 9-11 cycles of 15 sec separated by at least 90 sec of cooling. Cell debris was removed by centrifuging twice at 14,000 x g for 15 min at 4 °C. Protein concentration in the lysates was determined by BCA assay using BSA as a standard.  Immunoprecipitation was then conducted as follows: For a given ChIP timecourse, equal mass quantities (typically 1-1.5 mg) of lysate from each timepoint were prepared in 500 ul of lysis buffer each. For anti-RpaA ChIP, equal amounts (typically 10-15 µg) of affinity-purified antibody was added to each tube; for anti-HA ChIP, 40 ul (bed volume) of anti-HA agarose beads (Pierce) were added to each tube. Samples were overnight in the dark at 4 °C with continuous rotation. The following morning, for anti-RpaA ChIP only, 50-70 µl of rProtein A Sepharose Fast Flow beads (GE Healthcare; 33% slurry in lysis buffer) was added to each sample, and the samples were then incubated for 2 h at 4 °C with rotation. For both anti-RpaA and anti-HA ChIP, beads were isolated by centrifugation for 1 min at 1000 x g at room temperature. Beads were then washed twice with 1 ml lysis buffer, once with 1 ml of buffer B (50 mM HEPES pH 7.5, 500 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% sodium deoxycholate), once with 1 ml of wash buffer (10 mM TrisCl pH 8.0, 250 mM LiCl, 1 mM EDTA, 0.5% NP-40, 0.1% sodium deoxycholate) and finally with 1 ml of TE pH 7.5 (10 mM TrisCl pH 7.5, 1 mM EDTA); each wash was conducted for 5 min at room temperature on a tube rotator followed by isolation of beads by centrifugation for 1 min at 1000 x g at room temperature. Protein-DNA complexes were then eluted with 250 µl elution buffer (50 mM TrisCl pH 8.0, 10 mM EDTA, 1% SDS) for 1 h at 65 °C. For both this eluate and a matched sample of lysate not subjected to immunoprecipitation (“input DNA”), crosslinks were reversed for 6-18 h at 65 °C. Next, 250 ul of TE was added to each sample to dilute the SDS, followed by addition of 100 µg of proteinase K and 80 µg of glycogen. Samples were incubated for 2 h at 37 °C. Samples were then supplemented with 55 µl of 4 M LiCl and extracted with 1 ml of phenol/chloroform/isoamyl alcohol. DNA in the aqueous phase was precipitated with ethanol and then washed once with 80% ethanol. Pellets were air-dried and then resuspended in 50 µl of TE containing 20 ng/µl of DNase-free RNase (Fermentas) and incubated for 1 h at 37 °C. Samples were then supplemented with 150 µl of TE and 22.2 µl of 3 M sodium acetate pH 5.2, extracted with phenol/chloroform/isoamyl alcohol, and precipitated with ethanol; alternatively, DNA was purified using a Qiagen PCR purification kit. DNA concentration in ChIP samples was estimated either by PicoGreen assay (Invitrogen) or by qPCR based on the abundance of the coding region of synpcc7942_0612, a region showing no enrichment for RpaA binding in ChIP-Seq experiments; for both methods, input DNA quantified by absorption spectrophotometry was used as a standard. Except for Samples 9, 10, 11, 16, 17, and 19, libraries for Illumina sequencing of ChIP DNA were prepared following a protocol developed by Ethan Ford (http://ethanomics.files.wordpress.com/2012/09/chip_truseq.pdf), with modifications. 0.15-3 ng of ChIP DNA was used for each sample. DNA ends were blunted by treatment with 1.4 units of T4 DNA polymerase (NEB), 0.45 units of Klenow fragment (NEB), 4.5 units of T4 polynucleotide kinase (NEB), and 0.4 mM dNTPs (NEB) in 1x T4 DNA ligase buffer (NEB) in a total of 50 µl volume for 30 min at 20 °C. DNA was purified using 50 µl of AMPure XP beads (Beckman) and 50 µl of a solution containing 20% PEG8000 (Sigma) and 1.25 M NaCl. DNA was eluted in 16.5 ul of TE/10 (10 mM TrisCl pH 8.0, 0.1 mM EDTA). DNA was then A-tailed at the 3' ends by treating the eluate with 2.5 units of Klenow fragment lacking 3'-5' exonuclease activity (Klenow fragment 3'-->5' exo-, NEB) and 0.2 mM dATP (GE Healthcare) in 1x NEB Buffer 2 in a total volume of 20 µl for 30 min at 37 °C. TruSeq adapters (Illumina or Eurofins MWG Operon) were ligated onto the A-tailed DNA by addition of 25 µl of 2X Quick Ligase Buffer (NEB), 0.5 µl of ~250 nM TruSeq adapter (1:30 dilution of Illumina stock), 3 µl of nuclease-free ddH2O, and 1.5 µl of Quick Ligase (NEB) followed by incubation for 20 min at 21 °C. The ligation reaction was stopped by addition of 5 µL of 0.5 M EDTA pH 8.0 (Ambion). Next, DNA was purified using 55 µl of AMPure XP beads without additional PEG or salt. DNA was eluted in 15.5 µl of TE/10. The Y-shaped adapters were then linearized with 5 cycles of PCR (initial denaturation of 30 sec at 98 °C followed by 5 cycles of [10 sec at 98 °C, 30 sec at 60 °C, 30 sec at 72 °C] followed by 5 min at 72 °C) using Phusion polymerase (Thermo) in HF Buffer and 1 µl of TruSeq primers (25 µM) in a total volume of 31 µL. Linearized DNA was purified using 30 µl of AMPure XP beads without additional PEG or salt. DNA was eluted in 30 µl of TE/10. Fragments between 300 and 500 bp were size-selected using agarose gel purification and the QIAquick gel extraction kit (Qiagen) or using a Pippin Prep (SAGE Science). Purified DNA was further amplified with 13-14 cycles of PCR, as above, in a total volume of 62.5 µl. Following PCR, DNA was purified using 51 µl of AMPure XP beads without additional PEG or salt. DNA was eluted in 12 µl of TE/10. Libraries were assessed using a DNA High Sensitivity chip on an Agilent Bioanalyzer 2100. Samples were sequenced on an Illumina HiSeq instrument or Genome Analyzer II (Illumina) by the core facility at the Harvard FAS Center for Systems Biology.  Reads were aligned to the S. elongatus genome using Bowtie (Langmead et al., 2009), counting only those aligning uniquely to one location with up to three mismatches.  For Samples 9, 10, 11, 16, 17, and 19, libraries were prepared by Libraries for Solexa sequencing were prepared using paired-end adapters and PCR primers from Illumina following the manufacturer's protocol for ChIP-Seq sample preparation (part number 11257047 revision A) with modifications. All enzymes except for fast ligase were obtained from NEB instead of Illumina. First, 5 – 10 ng of ChIP or input DNA was blunted and phosphorylated with T4 DNA ligase, T4 DNA polymerase, and T4 polynucleotide kinase. 3' dA overhangs were added using exo- Klenow fragment. Adapters (diluted 1:10 from the Illumina stock) were ligated for 15 min at room temperature using LigaFast DNA ligase (Promega). Adapter-ligated fragments were size-selected on 2% agarose/TAE gel containing 400 μg/ml ethidium bromide; a gel slice corresponding to the 200-300 bp region (based on 50 bp ladder, Promega) was excised. DNA was purified from the gel using the Qiagen gel extraction kit according to the manufacturer's protocol, except that the gel slice was dissolved at room temperature. Adapter-ligated fragments were then amplified by PCR using PE Primers 1.0 and 2.0 (Illumina). The resulting libraries was purified with the Qiagen MinElute kit and eluted in 15 ul of buffer EB. The concentration and fragment length distribution of the libraries was then determined using the DNA 1000 Bioanalyzer assay (Agilent).
GEO Accession
GSM1238658