GSM4696248: MNaseChIP_Spt16_yFR930chd1D_R2; Saccharomyces cerevisiae; ChIP-Seq
MNase-ChIP experiments were performed in two biological replicates, as previously described (Rando, 2010) with minor modifications. Briefly, yeast cultures were grown in 500 mL of YPD to an OD600 of 0.7-0.9 before crosslinking with 1% formaldehyde (Fisher Scientific, BP531-500) at room temperature for 15 min and quenched with 125 mM glycine at room temperature for 5 min. Crosslinked cells were collected by centrifugation and washed twice with ice-cold ddH2O. Yeast cell wall digestion was then performed by resuspending cell pellets in 39 mL Buffer Z (1 M sorbitol and 50 mM Tris-HCl pH 7.4) containing 10 mM β-mercaptoethanol and 10 mg Zymolyase 100T (US Biological, Z1004) and incubating at 30˚C with agitation (20-40 min). When digestion was completed, the resulting spheroplasts were pelleted by centrifugation at 3,000 g for 5 min at 4°C and gently resuspended in 2.5 mL of NP buffer (1 M sorbitol, 50 mM NaCl, 10 mM Tris-HCl pH 7.4, 5 mM MgCl2, 1 mM CaCl2, 500 µM spermidine (Sigma-Aldrich, 85558), 1 mM β-mercaptoethanol and 0.075% NP-40) before being exposed to MNase (Worthington, LS004798) digestion titration to generate the appropriate size of chromatin fragments as follow. 700 µl of spheroplasts were incubated with 4.5, 9, 18, and 36 units of MNase (resuspended in 10 mM Tris-HCl pH 7.4 and 30% glycerol) at 37˚C for 20 min and reaction was stopped by adding 10 mM EDTA. To ensure IP conditions, 200 µL of adjusting buffer (50 mM HEPES-KOH pH7.5, 140 mM NaCl, 1% Triton X-100, and 0.1% Na-deoxycholate) were added to the digested chromatin sample. To determine the appropriate condition of MNase digestion, 45 µL (5%) of each MNase digested sample were taken and incubated with 5 μL of 10X TE/SDS at 65˚C for 16 hr to reverse crosslinking. Samples were then treated with RNase A (345 μL TE, 3 μL 10 mg/mL RNAse A, 2 μL 20 mg/mL Glycogen) at 37°C for 2 hr and subsequently subjected to Proteinase K (15 μL 10% SDS, 7.5 μL 20 mg/mL Proteinase K) digestion at 37°C for 2 hr. Samples were twice phenol/chloroform/isoamyl alcohol (25:24:1) extracted followed by precipitation with 200 mM NaCl and 100 % ethanol. Precipitated DNA was resuspended in 30 μL of TE, treated with 1µL of 10 mg/mL RNAse A at 37°C for 1 hr before being analyzed by electrophoresis on a 1.8% TAE-agarose gel stained with ethidium bromide. The digested samples were also qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA Kit (Agilent Technologies, 5067-4626). The condition giving approximately 65% mono-, 25% di- and 10% tri-nucleosomes was subjected to IP as follow. 45µL (5%) were saved as Input sample and the remaining digested chromatin was incubated overnight with the appropriate antibody-coupled Dynabeads as per our standard ChIP protocol (see above). Beads were then washed and samples reverse crosslinked, treated with RNase A, and subsequently subjected to Proteinase K before being twice phenol/chloroform/isoamyl alcohol extracted followed by precipitation with NaCl and ethanol. Precipitated DNA was resuspended in 50 μL of TE before being used in sequencing libraries.  Before proceeding with MNase-ChIP-seq libraries, ChIP and Input samples were subjected to a 2X cleanup using KAPA Pure Beads (Roche, 07983280001) according to the manufacturers' instructions. Samples were eluted with 50 μL of Elution buffer (10 mM Tris pH 8.0) and quantified/qualified on an Agilent 2100 Bioanalyzer instrument using the High Sensitivity DNA kit.  1 ng of Input DNA and 40 μL of ChIP DNA were used for library preparation as follow. The ends of DNA were repaired by incubating in 70 μL of 1X NEBuffer 2 containing 0.6 units of T4 DNA polymerase (NEB, M0203L), 2 units of T4 polynucleotide kinase (NEB, M0201S), 0.09 nM dNTPs and 0.045 μg/μL of BSA at 12˚C for 30 min. Repaired DNA was then subjected to a 2X cleanup using KAPA Pure Beads before dA-tailing as follow. Beads containing the repaired DNA were resuspended in 50 μL of 1X NEBuffer 2 containing 0.1 mM dATP and 25 units of Klenow Fragment (3'→5' exo-) (NEB, M0212M), and incubated at 37˚C for 30 min. After a 2X cleanup with KAPA Pure Beads, dA-tailed DNA was ligated to index adapters (Roche, SeqCap Adapter kit A (07141530001) and SeqCap Adapter kit B (07141548001)) as follow. The beads were resuspended in 45 μL of 1X Ligase buffer containing 8 nM of adapter and 2.5 units of T4 DNA ligase and incubated at room temperature for 60 min. The ligated DNA was then subjected to a 1X cleanup with KAPA Pure Beads. Libraries were PCR-amplified with 10-12 cycles using KOD Hot Start DNA polymerase (Millipore, 71086-3) and cleaned up using 1X KAPA Pure Beads. Libraries were qualified on Agilent 2100 Bioanalyzer using High Sensitivity DNA kit and quantified by qPCR using NEBNext Library Quant kit for Illumina (NEB, E7630). Equal molarity of each library was pooled (12 libraries per pool) and subjected to sequencing on Illumina HiSeq 4000 or NovaSeq 6000 platform at the McGill University and Génome Québec Innovation Centre to generate 50 bp paired-end reads. Barcode ID:Adapter 18 from SeqCap Adapter kits A and B (Roche, 07141530001 and 07141548001) MNase-ChIP-Seq