GSM2845174: ChIPseq_H3K27ac_NCC5-6ss_Citrine-Cherry; Danio rerio; ChIP-Seq
FACS sorted cells were washed with PBS and stored at -80C in lysis buffer. RNA was extracted using Ambion RNAqueous Micro Total RNA isolation kit (AM1931), checked on Bioanalyser, samples with RIN >7 were used to prepare cDNA using Takara Clontech SmartSeq2 V4 kit (634889). Sequencing libraries were prepared using Illumina Nextera XT library preparation kit (FC-131-1024). For histone ChIP, FACS sorted cells were cross-linked with 1% formaldehyde and sonicated to 300-800bp fragments. Preblocked Protein A Dynabeads were incubated with antibody (Abcam Ab4729). Sonicated DNA-protein complexes were applied to beads, IgG antibody (Millipore 12-370) was used as control and an input sample was taken. Samples were washed, cross-links were reversed, immunoprecipitated DNA was eluted, purified and libraries prepared using amplification protocol for small cell numbers (Adli and Bernstein, 2011). H3K27Ac ChIP libraries were sequences using 50bp paired end reads using Illumina Hiseq2500 platform.