title: GSM1977386: inHA_II; Ustilago maydis; ChIP-Seq
library_construction_protocol: Samples were ground to a fine powder in liquid nitrogen. For chromatin preparation, powder was resuspended in lysis buffer (50mM HEPES  pH 7.5, 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 5 mM benzamidine, 2 mM PMSF, 1 X Roche complete EDTA free protease inhibitors) and further treated by sonication to lyse the remaining cells using a microtip sonifier (Branson). Chromatin was then sheared in a bioruptor sonication bath (Diagenode) for 10 cycles (30s on / 30s off) at high power setting. An aliquot was saved to serve as input control and the rest of the chromatin solution was incubated with ChIP grade Protein A/G magnetic beads (Life technologies) coupled to a monoclonal anti-HA antibody (Sigma) for 10h at 4°C. Beads were washed three times in lysis buffer, two times in high salt buffer (50 mM HEPES pH 7.5, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 1 X Roche complete EDTA free protease inhibitors) and once in TE buffer (10 mM Tris-HCl, 1 mM EDTA, pH 7.5). DNA / Ros1HA complexes were eluted from the beads in TE SDS buffer (10 mM Tris-HCl, 1 mM EDTA, 1% SDS, pH 7.5) by incubation for15 min at 65°C. Samples and input controls were de-crosslinked for 8-10h at 65°C in TE SDS buffer containing 200 mM NaCl and 0.65 µg/µL proteinase K. DNA was purified using the ChIP DNA clean and concentrator kit (Zymo research). The experiment was done in three biological replicates which were sequenced separately. DNA libraries were prepared using the Illumina TruSeq RNA sample preparation Kit v2 starting from the end repair step of the protocol. Up to 100 ng ChIP DNA was used as starting material. After end repair and A-tailing, indexing adapters were ligated. The products were then purified and amplified for 18 PCR cycles to create the final libraries. After validation (Agilent 2200 TapeStation) and quantification using the KAPA Library Quantification Kit (VWR, Erlangen, Germany) and the Applied Biosystems 7900HT Sequence Detection System, equimolar amounts of library were pooled. The pool was sequenced using the Illumina TruSeq PE Cluster Kit v3 and the Illumina TruSeq SBS Kit v3-HS (101x7x101 Cycles) on an Illumina HiSeq 2000 sequencer with a paired-end (101x7x101 cycles) protocol.
attributes.tag: GEO Accession
attributes.value: GSM1977386