GSM4598798: Input; Homo sapiens; ChIP-Seq
Lysates were fragmented by Mnase digestion and histone-DNA complexes were isolated with antibody. ChIP samples were purified using Agencourt AMPure XP beads (Beckman Coulter) and quantified with the Qubit (Invitrogen). For the H3K122succ samples and respective input, the ChIP-seq libraries were prepared from 5 to 10 ng of double-stranded purified DNA using the MicroPlex Library Preparation kit v2 (C05010014, Diagenode s.a., Seraing, Belgium), according to manufacturer's instructions. While for H3K4me3, H3K9ac, H3K27ac and respective input, the ChIP-seq libraries were prepared from 5 ng of double-stranded purified DNA using the NEBNextâ Ultra II (E7645S, New England Biolabs). 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 (8 cycles). Amplified libraries were purified and size-selected using Agencourt AMPure XP beads (Beckman Coulter) to remove unincorporated primers and other reagents.