GSM6333341: Rep2, BF, PHD6, IP; Trypanosoma brucei brucei; ChIP-Seq
3-5 x 10^8 cells were harvested and fixed with 0.8% formaldehyde (final concentration) for 20 min at room temperature. Cells were lysed and sonicated for 30 cycles (30 s on, 30 s off; high setting) in a Bioruptor sonicator (Diagenode) in the presence of 0.2% SDS. Inputs (0.8% of total volume/sample) were taken before incubating the rest of the cell lysates overnight with 10 µg anti-GFP antibody (A-11122, Invitrogen) and Protein G Dynabeads. The beads were washed first in the presence of 500 mM NaCl, and then in the presence of 250 mM LiCl. The DNA was eluted from the beads with 1% SDS and treated with RNAse and Proteinase K. Finally, the DNA was purified using a QIAquick PCR Purification Kit (Qiagen). Immunoprecipitated and input DNA was blunt ended using a Quick Blunting Kit (NEB). DNA was then purified using AMPure beads (Beckman Coulter) with a beads:DNA ratio of 1.6:1 (v/v). DNA was A-tailed using Klenow Fragment exo (NEB) and NEXTflex adapters (Bioo Scientific) were ligated to the DNA fragments using a Quick Ligation Kit (NEB). Samples were purified twice with AMPure beads:DNA ratio of 1:1 (v/v), and DNA was then amplified by PCR (14-18 cycles). Following PCR, DNA was purified with AMPure beads:DNA ratio of 0.65:1 (v/v), and the resulting supernatant was taken for a 0.7:1 bead:sample purification (v/v). Finally, two 1:1 (v/v) DNA:AMPure beads purifications were performed. The DNA amount in each library was quantified using Qubit dsDNA HS Assay Kit (Invitrogen). Fragment size distribution in each library was assessed using a Bioanalyzer High Sensitivity DNA Analysis kit (Agilent).