ERX193140 SAMEA2045034 Glutamine_methylation_on_Histone_H2A_is_an_RNA_Polymerase_I_dedicated_modification ChIP-Seq GENOMIC ChIP xxx xxx xxx xxx Illumina_MiSeq ERA188727 ERS205786 ERP002121 PRJEB1290 2014-08-12T17:59:07Z Organism taxonomy_id="4932" taxonomy_name="Saccharomyces cerevisiae" Alias=E-MTAB-1447:H2A Broker name=ArrayExpress Description=Protocols: Yeast cells were grown to mid-logarithmic phase before fixation 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. Samples were purified with the Qiagen PCR purification kit following manufacturer's guidelines. INSDC center alias=University of Cambridge INSDC center name=University of Cambridge INSDC first public=2014-01-21T17:01:05Z INSDC last update=2018-03-08T16:12:57Z INSDC status=public SRA accession=ERS205786 Sample Name=ERS205786 Title=H2A age=2 year biosource provider=Takehido Kobayashi biosource type=frozen sample genotype=Mata ade2-1 ura3-1 his3-11 trp1-1 leu2-3,112 can1-100 fob1D::LEU2 (20 rDNA copies) strain or line=YSI1102