GSM8031906: TBP in KO; Mus musculus; ChIP-Seq
Cells were fixed with 0,4 % PFA for sonicated chromatin and with 1% PFA for MNase digested chromatin. Cells were sonicated on E220 Focused-ultrasonicator (Covaris) either MNase digested according protocol described in (Chromatin Immunoprecipitation Assay Using Micrococcal Nucleases in Mammalian Cells. T. Yamakawa , K. Itakura. J Vis Exp. 2019 May 10:(147). doi: 10.3791/59375). Precipitated chromatin was de cross-linked and DNA was purified with phenol-chloroform. ChIP samples were purified using SPRIselect beads (Beckman-Coulter, Villepinte, France) and quantified using the Qubit 4 fluorimeter (Thermo Fischer Scientific, Illkirch, France). ChIP-seq libraries were prepared from 0.1 to 7 ng of double-stranded purified DNA using the MicroPlex Library Preparation kit v3 (C05010001, Diagenode, Seraing, Belgium), according to manufacturer's instructions. In the first step, the DNA was repaired and yielded molecules with blunt ends. In the next step, stem-loop adaptors with blocked 5 prime ends were ligated to the 5 prime end of the genomic DNA, leaving a nick at the 3 prime end. The adaptors cannot ligate to each other and do not have single-strand tails, avoiding non-specific background. In the final step, the 3 prime ends of the genomic DNA were extended to complete library synthesis and Illumina compatible indexes were added through a PCR amplification (10 + 4 cycles). Amplified libraries were purified and size-selected using SPRIselect beads (Beckman Coulter) to remove unincorporated primers and other reagents.