GSM2629874: Set2-ChIPseq; Saccharomyces cerevisiae; ChIP-Seq
Cells were collected by centrifugation, washed with 1 × TBS (20 mM Tris-HCL at pH 7.5, 150 mM NaCl) and twice with lysis buffer (50 mM HEPES-KOH at pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X100, 0.1% Na deoxycholate, 0.1% SDS). Cell pellets were resuspended in 2 mL lysis buffer and lysed by bead beating and the lysate was sonicated in a Covaris S220 system. Chromatin was washed with FA lysis buffer and solubilized via sonication with a Covaris S220 instrument (COVARIS, INC.) to yield an average DNA fragment size of 200 bp as determined on an Agilent 2200 TapeStation instrument (see below). This was achieved by sonicating the sample for 18 minutes using the following parameters: Peak Incident Power (W): 140; Duty Factor: 5%; Cycles per Burst: 200. 30 µL and 50 µL of the washed and fragmented chromatin samples were saved as input materials and for control of the average chromatin fragment size, respectively. The remaining chromatin sample was immunoprecipitated with 100 μL antibody-coated and prewashed magnetic Dynabeads® Protein G (life technologies) at 4 °C for 3 hr on a turning wheel. Immunoprecipitated chromatin was washed 5 times with ChIP wash buffer (100 mM Tris-HCl at pH 7.5, 500 mM LiCl, 1% NP-40, 1% Na deoxycholate) and one time with TE buffer (10 mM Tris-HCl at pH 7.5, 1 mM EDTA). Immunoprecipitated chromatin was eluted for 10 minutes at 70 °C in the presence of ChIP elution buffer (100 mM Sodium bicarbonate, 1% SDS). Eluted immunoprecipitated chromatin as well as input material and material for control of the average chromatin fragment size were incubated with 10 µL RNase A (10 mg/mL) at 37 °C for 30 minutes and subsequently subjected to Proteinase K (20 µL of 20 mg/mL Proteinase K, Bioline, BIO-37084) digestion at 37 °C for 2 hr and reversal of crosslinks (at 65 °C over night). DNA of the IP, mock IP, input, and fragment size control samples was purified with the QIAquick MinElute PCR Purification Kit (Qiagen) according to the manufacturers’ instructions. Samples used for determining the average chromatin fragment size were analyzed on an Agilent 2200 TapeStation instrument. DNA concentration of the IP and Input samples was determined with Qubit 1.0, dsDNA HS. Before preparing libraries for Illumina sequencing, IP and input samples were analysed via qRT-PCR on 4 housekeeping genes to access quality of sample DNA. To prepare libraries of ChIP samples, 1–10 ng of IP or Input DNA were used for sequencing library preparation according to the manufacturer's recommendations using the ThruPLEX® DNA-seq Kit. Libraries were qualified on an Agilent 2200 TapeStation instrument and quantified with Qubit 1.0. Libraries were pooled and sequenced on an Illumina HiSeq 2500 sequencer.
GEO
GSM2629874