title: GSM1256611: Smc6-myc, spo11delta, 4.5h in SPM; Saccharomyces cerevisiae SK1; ChIP-Seq
library_construction_protocol: ChIP from meiotic cultures was performed as described by Panizza (2011). In brief, 4x10^9 cells were collected per time point and incubated with para-formaldehyde (1% final concentration) for 15 minutes at 25°C. Cross-linking was stopped by addition of glycine to 131 mM. 4x10^9 cells were divided into 8 aliquots and separately opened using a multibead shocker (YASUI-KIKAI, Osaka) at 2,500 rpm, 15 cycles of 30 sec on and 30 sec off, at 4°C. Extracts were sonicated to an average of 200bp by a Covaris S2 instrument. After removing the cell debris, the supernatant was used for the chromatin immunoprecipitation. For each sample, 50 μl Dynabeads Pan mouse IgG (Invitrogen), 50 μl Dynabeads® Pan Mouse IgG (invitrogen) were incubated with the 9E11 anti-myc (mouse) antibody for 6-15 hr at 4°C. The precipitated DNA was used as a template for quantitative real-time PCR using GoTaq qPCR Master Mix for SYBR assay (Promega) (primer sequences are available upon request). For genome-wide analysis (ChIP-seq), one-tenth of the ChIP DNA was analyzed by real-time PCR (qChIP) while the remaining DNA was concentrated by precipitation in ethanol. For the preparation of libraries for Illumina sequencing, we strictly followed the protocols provided by Illumina (Illumina ChIP-seq DNA sample prep protocol). Briefly, DNA-ends were repaired to convert overhangs to phosphorylated blunt ends with T4 DNA polymerase, E. coli DNA Pol I (Klenow fragment), and T4 polynucleotide kinase (PNK). An 'A' nucleotide was added to the 3' end of the blunt ended fragments using Klenow fragment (3' to 5' exo minus). This prepared the DNA fragments for ligation to the adapters (Truseq Adaptor1-5) which have a single 'T' base overhang at their 3' ends. After ligation, excess adaptors were removed by selecting a certain size range from 200-500bp with QIAquick Gel Extraction Kit (Qiagen). Purified templates were PCR amplified with 15-18 cycles by KAPA-HiFi Hot Start PCR Kit (KAPA biosystems). Before hybridization to the flow cell, the amount and the size of the DNA library was controlled to be at least 1µg/ul (for an average fragment size of 300bp).
attributes.tag: GEO Accession
attributes.value: GSM1256611