GSM9207351: 3-day_untr_AtfA_ChIP-seq_rep1; Aspergillus nidulans; ChIP-Seq
2 d and 3 d old untreated and 3d old 0.04 mM MSB-treated conidia were crosslinked with freshly prepared 1% formaldehyde at room temperature for 30 min. Following that, 1/20 volume of 2.5 M glycine solution was added to stop the cross-linking reaction. Conidia were then collected by centrifugation (5000 g, 5 min, 4 oC) and washed with ice-cold PBS three times. About 1.5 ml FA lysis buffer {50 mM HEPES-KOH [pH 7.5], 150 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% Na deoxycholate, 0.1% SDS, 1 protease inhibitor cocktail tablet (Roche) per 50 ml} was added before use. Glass beads were added into cross-linked cell lysates, which were broken by a mini-bead beater (MagNa Lyser instrument, Roche, Switzerland) for three cycles (1 min homogenization followed by 1.5 min resting on ice). Subsequently the samples were sonicated for eight cycles (60 s on, 60 s off) with a sonifier level 5 of output control in a Diagenode Bioruptor (Diagenode, USA). All steps were carried out at 4 oC. The sonicated cell lysates were cleared of cellular debris by centrifugation at 13 000 rpm for 10 mins at 4 oC. The supernatant was collected and subjected to chromatin IP with mouse monoclonal Anti-Flag M2 antibody (Merck, USA) using MAGnify™ Chromatin Immunoprecipitation System (Invitrogen, USA) following the manufacturer's protocol. ChIP-Seq libraries were prepared with Ovation Ultralow Library Systems V2 (Tecan, Switzerland) according to the manufacturer's instructions. Briefly, 10 ng of immunoprecipitated DNA was used for library generation. Repairing end step was followed by indexed adaptor ligation and enrichment of adaptor ligated fragments by 10 PCR cycles. Fragment size distribution of the libraries was check on BioAnalyzer 2100 (Agilent Technologies, USA) using DNA High Sensitivity chip. Single read 75 bp sequencing reads were generated by NextSeq 500 sequencer (Illumina, USA). Library preparations and sequencing were performed by the Genomic Medicine and Bioinformatics Core Facility of the University of Debrecen.