GSM5068211: Rep2_DOT1A_input; Trypanosoma brucei brucei; ChIP-Seq
Briefly, 4 x 10^8 parasites were harvested and fixed with 0.8% formaldehyde for 20 min at room temperature. Cells were lysed and sonicated in the presence of 0.2% SDS for 30 cycles (30 s on, 30 s off) using the high setting on a Bioruptor sonicator (Diagenode). Input samples were taken before incubating the rest of the cell lysates overnight with 10 µg rabbit anti-GFP antibody (Thermo Fisher Scientific) and Protein G Dynabeads. The beads were washed and the DNA eluted from them was treated with RNAse and Proteinase K. DNA was purified using a QIAquick PCR Purification Kit (Qiagen). ChIP-seq libraries were constructed using 0.5-20 ng of immunoprecipitated or input DNA. Following blunt-ending and poly(A)-tailing, NEXTflex (Bioo Scientific) adapters containing barcodes were ligated to each DNA fragment. Fragments were size selected using AMPure beads (Beckman Coulter). The amount of DNA in the resulting libraries was quantified by Qubit fluorometer using Qubit dsDNA HS Assay Kit (Invitrogen). Fragment size distribution was assessed by 2100 Bioanalyzer Instrument (Agilent Technologies) using a Bioanalyzer High Sensitivity DNA Analysis kit (Agilent). Ideally, the libraries had a peak at 300 bp (~ 150 bp fragment DNA + ~ 150 bp adapter sequence). Libraries were sequenced on Illumina MiniSeq, HiSeq 4000 or NextSeq 550. In all cases, 75 bp paired-end sequencing was performed.