7 protocols
Called reads from the Illumina MiSeq system were mapped to the latest version of the Saccharomyces cerevisiae genome (sacCer3) using the Burrows Wheeler Aligner (BWA) with parameters -n 10 -k 3 -l 20
nucleic acid library construction protocol
50% of a ChIP reaction was used to generate the library. Library production followed the protocol published on http://ethanomics.wordpress.com/chip-seq-library-construction-using-the-illumina-truseq-adapters/ (2012) with few changes. Purifications steps using AMPure XP bead were replaced by the ChIP DNA Clean & Concentrator kit by Zymoresearch. Illumina adaptors were exchanged for Bioo Nextflex adaptors 1-3 (1:100 dilution each). Size selection was performed on a SAGE Pippin Prep using 2% gels and selecting for a size between 350-600 bp. The quality of the library was assessed using Agilent DNA High Sensitivity ChIPs and Qiagen Qubit.
Samples were purified with the Qiagen PCR purification kit following manufacturer's guidelines.
Cells were fixed with 1% formaldehyde for 45 minutes at room temperature, washed with PBS and resuspended in 500ul of SDS lysis buffer (1% SDS, 10mM EDTA, 50mM Tris HCl (8.0), 0.5mM PMSF-EtOH, 0.8_g/ml pepstatin A, 0.6_g/ml leupeptin). Cells were lysed by bead-beating 4x 1min with 3min on ice in between. Cell debris was removed by centrifugation and lysates were sonified for 30min in a constantly water-cooled Bioruptor. Sonified lysates were centrifuged and the supernatant was diluted 1:10 into IP-buffer (0.01% SDS, 1.1%Triton-X-100, 1.2mM EDTA, 16.7MM Tris HCl (8.0), 167mM NaCl, 0.5mM PMSF, 0.8_g/ml Pepstatin A, 0.6_g/ml leupeptin). Antibodies were added (1ug each of IgG (ab37415), H2A (Active Motif 39235) and 1/500 dilution of H2AQ105me) over night. Next day, 30ul of a Protein A/G Dynabead mix was added and beads were incubated for 1h. Subsequently, beads were washed with 1ml each TSE150 (1% Triton-X-100, 0.1%SDS, 2mM EDTA, 20mM Tris HCl (8.0), 150mM NaCl), TSE500 (1% Triton-X-100, 0.1%SDS, 2mM EDTA, 20mM Tris HCl (8.0), 500mM NaCl), and LiCl-buffer (0.25M LiCl, 1% NP-40, 1% dioxycholate, 1mM EDTA, 10mM Tris HCl (pH8.0)). Finally, beads were wahed with 1ml TE and eluted with elution buffer (1% SDS, 0.1M NaHCO3) for 30min at room temperature. 500mM NaCL (final) was added and crosslinks were reversed at 95oC for 30min. Samples were treated with 50ug RNase/DNase free.
Yeast cells were grown to mid-logarithmic phase before fixation
The amplified DNA was used for cluster generation on the Illumina MiSeq following manufacturer's instructions.
Libraries were pooled and sequenced on a Illumina MiSeq using single-ended 50bp readsSamples were amplified