At 25, or 32 min after release, cell aliquots were fixed with 1% (w/v) formaldehyde for 15 min at 30°C followed by 5 min of quenching in 125 mM Glycine. Cell pellets were then washed with cold PBS and flash frozen in liquid nitrogen and kept at -80°C until further processing.
Growth protocol
Cells were grown overnight at 30°C in SCD-URA. The culture was diluted to OD600 ~0.3 the next morning and grown to OD600 ~0.65 and re-diluted to OD600 ~0.3 in fresh media. The culture was synchronized with the addition of 0.15 μg/ml α factor for 3h30min at 30°C. Cells were released from arrest into preheated (SCD-URA) + 10 μM EdU.
Extracted molecule
genomic DNA
Extraction protocol
Cross-linked frozen cell pellets were re-suspended in 1.5 ml NP lysis buffer (100 mM NaCl, 10 mM Tris 7.4, 5 mM MgCl2, 1 mM CaCl2, 10% NP-40, 50 mM EDTA, 0.1 % SDS (optional), 1 mM PMSF and 1xEDTA-free protease inhibitor cocktail (Roche)). The suspension was then split into aliquots each containing ~billion cells. Zirconium Sillicate beads (400 μl, 0.5 mm) were then added to each aliquot and cells were mechanically disrupted using a bullet blender (Next Advance) for 4 times x 3 min (intensity 8). Zirconium beads were removed from the cell lysate by centrifugation and the entire cell lysate was subject to sonication using the Bioruptor-Pico (Diagenode) for 3x10 cycles of 30 seconds ON/OFF each resulting in a final median size of chromatin fragments of 200 bp. Cellular debris was then removed by centrifugation and 2 % of the total supernatant volume was kept for input fractions.The remainder of the sonicated chromatin was precleared using Protein A agarose beads (Repligen) for 1 hour (1h) at 4°C on the rotating wheel. The sonicated material was then pooled together and distributed into 500 µL aliquots (equivalent of 700 million cells per aliquot) and 25 µl of Protein G magnetic beads (Life Technologies-Invitrogen) pre-bound with 6µg of anti-HA (ab9110, abcam), was added to each tube. Aliquots were then incubated with rotation at 4°C overnight. The beads were then washed once with cold buffer L (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate), three times with cold Buffer W1 (Buffer L with 500mM NaCl), twice with cold Buffer W2 (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA), and once with cold TE buffer (10 mM Tris-HCl,pH 8.0, 1 mM EDTA). Chromatin was eluted in 2x125 μl elution buffer (25mM Tris-HCl pH 7.5, 5 mM EDTA, 0.5% SDS) after incubation for 2x10 min at 65°C. The eluates and reserved input samples were treated with RNase A (Qiagen) for 1h in 37°C and proteins were then digested with Proteinase K (Euromedex, final concentration 0.4 mg/ml) for 2h at 37°C and the temperature was then shifted to 65°C overnight to reverse cross-links. DNA was then purified with the QIAquick PCR purification kit (QIAGEN)
Label
Cy3
Label protocol
ChIPed DNA samples and their corresponding input samples were amplified, with a starting amount of up to 30 ng, using the DNA linear amplification method described previously (Liu et al., 2005; Liu et al., 2003). 2.5 µg of aRNA from each sample produced from the linear amplification was transformed into cDNA by reverse transcription in the presence of amino-allyl dUTP. The resulting cDNA was dye-coupled with Cy5 or Cy3 NHS-esters and purified as described previously (Liu et al., 2005).
Channel 2
Source name
sonicated chromatin from S-phase 32min after release from G1 arrest
At 25, or 32 min after release, cell aliquots were fixed with 1% (w/v) formaldehyde for 15 min at 30°C followed by 5 min of quenching in 125 mM Glycine. Cell pellets were then washed with cold PBS and flash frozen in liquid nitrogen and kept at -80°C until further processing.
Growth protocol
Cells were grown overnight at 30°C in SCD-URA. The culture was diluted to OD600 ~0.3 the next morning and grown to OD600 ~0.65 and re-diluted to OD600 ~0.3 in fresh media. The culture was synchronized with the addition of 0.15 μg/ml α factor for 3h30min at 30°C. Cells were released from arrest into preheated (SCD-URA) + 10 μM EdU.
Extracted molecule
genomic DNA
Extraction protocol
Cross-linked frozen cell pellets were re-suspended in 1.5 ml NP lysis buffer (100 mM NaCl, 10 mM Tris 7.4, 5 mM MgCl2, 1 mM CaCl2, 10% NP-40, 50 mM EDTA, 0.1 % SDS (optional), 1 mM PMSF and 1xEDTA-free protease inhibitor cocktail (Roche)). The suspension was then split into aliquots each containing ~billion cells. Zirconium Sillicate beads (400 μl, 0.5 mm) were then added to each aliquot and cells were mechanically disrupted using a bullet blender (Next Advance) for 4 times x 3 min (intensity 8). Zirconium beads were removed from the cell lysate by centrifugation and the entire cell lysate was subject to sonication using the Bioruptor-Pico (Diagenode) for 3x10 cycles of 30 seconds ON/OFF each resulting in a final median size of chromatin fragments of 200 bp. Cellular debris was then removed by centrifugation and 2 % of the total supernatant volume was kept for input fractions.The remainder of the sonicated chromatin was precleared using Protein A agarose beads (Repligen) for 1 hour (1h) at 4°C on the rotating wheel. The sonicated material was then pooled together and distributed into 500 µL aliquots (equivalent of 700 million cells per aliquot) and 25 µl of Protein G magnetic beads (Life Technologies-Invitrogen) pre-bound with 6µg of anti-HA (ab9110, abcam), was added to each tube. Aliquots were then incubated with rotation at 4°C overnight. The beads were then washed once with cold buffer L (50 mM HEPES-KOH, pH 7.5, 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na-deoxycholate), three times with cold Buffer W1 (Buffer L with 500mM NaCl), twice with cold Buffer W2 (10 mM Tris-HCl, pH 8.0, 250 mM LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, 1 mM EDTA), and once with cold TE buffer (10 mM Tris-HCl,pH 8.0, 1 mM EDTA). Chromatin was eluted in 2x125 μl elution buffer (25mM Tris-HCl pH 7.5, 5 mM EDTA, 0.5% SDS) after incubation for 2x10 min at 65°C. The eluates and reserved input samples were treated with RNase A (Qiagen) for 1h in 37°C and proteins were then digested with Proteinase K (Euromedex, final concentration 0.4 mg/ml) for 2h at 37°C and the temperature was then shifted to 65°C overnight to reverse cross-links. DNA was then purified with the QIAquick PCR purification kit (QIAGEN)
Label
Cy5
Label protocol
ChIPed DNA samples and their corresponding input samples were amplified, with a starting amount of up to 30 ng, using the DNA linear amplification method described previously (Liu et al., 2005; Liu et al., 2003). 2.5 µg of aRNA from each sample produced from the linear amplification was transformed into cDNA by reverse transcription in the presence of amino-allyl dUTP. The resulting cDNA was dye-coupled with Cy5 or Cy3 NHS-esters and purified as described previously (Liu et al., 2005).
Hybridization protocol
Labeled probes (a mixture of Cy3 labeled Rpb3-HA_ChIP_DNA|sonicated_chromatin_genomic DNA and Cy5sonicated_chromatin_genomic DNA|labeled Rpb3-HA_ChIP_DNA) were hybridized onto an Agilent yeast 4x44K whole genome DNA array (ref. G4810A-14810).
Scan protocol
Fluorescent array images were collected for both Cy3 and Cy5 with an InnoScan 710 MicroArray scanner (Innopsys) at 5 micron resolution and processed with the Mapix software.
Data processing
After background correction and removal of flagged values, data was block normalized by dilation and log base 2 ratios (Cy3/Cy5) were calculated to produce the values given in the data table.
The Asymmetric Distribution of RNAPII and Nucleosomes on Replicated Daughter Genomes is caused by Differences in Replication Timing between the Lagging and the Leading Strand [ChIP]
The Asymmetric Distribution of RNAPII and Nucleosomes on Replicated Daughter Genomes is caused by Differences in Replication Timing between the Lagging and the Leading Strand
