Illumina HiSeq 2000 sequencing; H3K3 me1 and H3K4me3 ChIP-seq in Yeast
H3K3 me1 and H3K4me3 ChIP-seq in Yeast
Yeast cells were collected by centrifugation 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media Strain YPH499 (Mata ura3-52 lys2-801 ade2-101 trp1-Δ63 his3-Δ200 leu2-Δ1) was transformed with 2, 3 or 4 expression plasmids by the standard lithium acetate procedure. Transformants were selected on plates of appropriate selective medium with 2% Raffinose and 10µg/ml doxycycline to repress any expression. Selected transformants (2 to 4 transformants per combination of plasmids) were grown on selective liquid medium with 2% Raffinose and 10µg/ml doxycycline up to OD600=0.5. Then, yeast cells were spun 10min at 1000x g, washed twice with sterilized water, and resuspended into selective media with 1% Raffinose and 2% Galactose without doxycycline to allow expression of all four DNMTs. For experiments on synchronized cells, cells were treated with alpha-factor (3µM final) for 2 to 4 hours to synchronise cells in G1 DNA was purified using phenol–chloroform extraction and concentrated by ethanol precipitation. Each chromatin extract was then used as an input control and for two separate immunoprecipitations (H3K4me1 and H4K4me3), thus INE1 is the input both for the M1E1 and M3E1, INE2 for the M1E2 and M3E2, INM1 for M1M1 and M3M1 and INM2 for M1M2 and M3M2. After crosslinking, spheroplasts were isolated as described before using zymolase and resuspended in 0.3 ml of lysis buffer (50 mM Hepes-KOH at pH 7.2, 140 mM NaCl, 1 mM EDTA, 0.1% Deoxycholic acid sodium salt and 1% Triton X-100) containing a cocktail of protease inhibitors (EDTA-free Tablets, Roche). An equal volume of glass beads (0.5-mm diameter) was added, and the spheroplasts were broken using a bead-beater (FastPrep-24, Biomedicals). Glass beads were then removed and the lysate was transferred to a Sorenson tubes to digest the chromatin into fragments of 300 nucleotides using the Bioruptor Pico (30 cycles, 30”on/30”off). The whole extract was clarified by centrifugation for 10 min at 5000 × g at 4 °C and an aliquot was taken as input. In parallel, 50 µl of Dynabeads M-280 Sheep anti Rabbit IgG (Thermo Fisher) per sample were washed twice with PBS+5mg/ml of BSA and incubated overnight at 4ºC with 2.5 µg of the primary antibody (H3K4me1 (ab8895, Abcam) or H3K4me3 (ab8580, Abcam)). Beads were then washed with PBS+5mg/ml of BSA and resuspended in 30 µl/sample of PBS-BSA 5mg/ml. Extracts were then incubated 2h at 4ºC with the Dynabeads, previously conjugated with the primary antibody, and then washed two times with lysis buffer, two times with the lysis buffer supplemented with 360mM NaCl,, 2 times with washing buffer (0.5% Deoxycholic acid sodium salt, 10mM TRIS pH8, 250 mM LiCl, 0.5% NP-40, 1 mM EDTA) and one time with TE (10Mm Tris-HCl pH 7.5,1mM EDTA). Dynabeads were resuspended in 80 µl of Elution Buffer (50 mM TRIS, 10 mM of EDTA, 1% SDS) and crosslinking was reverted incubating overnight at 65ºC followed by 2h incubation at 37ºC with 0.80 mg/ml per sample of proteinase K.