7 protocols
AccessionNameType
P-MTAB-30499
bioassay_data_transformation
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
P-MTAB-30500
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.
P-MTAB-30496
nucleic_acid_extraction
Samples were purified with the Qiagen PCR purification kit following manufacturer's guidelines.
P-MTAB-30495
specified_biomaterial_action
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.
P-MTAB-30494
grow
Yeast cells were grown to mid-logarithmic phase before fixation
P-MTAB-30498
scanning
The amplified DNA was used for cluster generation on the Illumina MiSeq following manufacturer's instructions.
P-MTAB-30497
sequencing
Libraries were pooled and sequenced on a Illumina MiSeq using single-ended 50bp readsSamples were amplified