GSM3357440: Scc1 ChIP_0; Trypanosoma brucei brucei; ChIP-Seq
300 million cells were harvested by centrifugation and fixed in the presence of 1% formaldehyde. Cells were washed and permeabilized with digitonin and chromatin sheared by sonication. An input sample was seperated from the chromatin and stored at -20 °C. The IP was performed by addition of Ig-coated magnetic beads (BB2 Antibody against the Ty1-tag) and incubation over night at 4°C whilst rotating. The DNA was subsequently washed and eluted from the beads. Crosslinks were reversed in both the ChIP and input sample. The samples were purified and the concentration was determined using Qubit. ChIP and input samples were end-repaired using T4 DNA polymerase (NEB), Klenow large Fragment (NEB) and T4 DNA Polynucleotide Kinase (NEB). Protruding A-bases were added to the 3` end using  Klenow fragment (3´ to 5´ exo minus) and dATP. TruSeq adapters were ligated to the cDNA fragments using Quick Ligase (NEB) and was purified using AMPure XP magnetic beads. Y-shaped adapters were converted to dsDNA with Illumina primers for 5 cycles, purified with magnetic beads  and library fragments of 200-500 bp were size selected from a 6% PAGE gel. The gel-purified library was further amplified for 10 cycles using Illumina primers and purified using magnetic beads. Libraries were quantified using Qubit and qPCR and were sequenced on an Illumina flow cell in an Illumina NextSeq apparatus following the manufacturer's instructions.
GEO
GSM3357440