Illumina HiSeq 2000 paired end sequencing; Nucleosome positioning in Yeast expressing active or inactive murine DNMTs
Nucleosome positioning in Yeast expressing active or inactive murine DNMTs
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. The short-insert paired-end libraries for MNase sequencing were prepared with PCR free protocol using KAPA Library Preparation kit (Roche). In short, 2.0 micrograms of Micrococcal nuclease (MNase) digested genomic DNA from S. cerevisiae was end-repaired, adenylated and ligated to Illumina platform compatible adaptors with dual indexes (Integrated DNA Technologies). The adaptor-modified end library was size selected and purified with AMPure XP beads (Agencourt, Beckman Coulter). The final libraries were quantified by Kapa Library Quantification Kit for Illumina platforms (Roche).
Institute for Research in Biomedicine (IRB Barcelona)-The Barcelona Institute of Science and Technology.
E-MTAB-10254:AP2775_p