GSM3071209: top-2 nmad-1 deletion flag L4 IP rep2; Caenorhabditis elegans; ChIP-Seq
L4 stage worms were collected at 20°C. The worms were crosslinked with 1x linking buffer (11 mM HEPES-NaOH, 110 mM NaCl, 1.1 mM EDTA and 1.1 mM EGTA) with 1% formaldehyde for 10 minutes at RT. The reaction was quenched by adding glycine to 0.125 M final concentration for 5 minutes at RT. Samples were washed with cold PBS twice. The samples were next dissociated with lysis buffer and sonicated by biorupter for 25 cycles in the cold room with 30 seconds on and 30 seconds off per cycle. Protein G beads were coupled to the flag antibody (F3165 Sigma) for 2 hours. Uncoupled Protein G beads were incubated with the sonicated samples to pull down non-specific binding chromatin. The precleared chromatin samples were incubated with antibody-coupled bead slurries and rotated at 4°C overnight. The bound DNA was eluted from the beads by 200  µl TE with 1% SDS after washing with low salt (0.2% SDS, 2% Triton X-100, 4 mM EDTA, 40 mM Tris-HCl), high salt (0.2% SDS, 2% Triton X-100, 4 mM EDTA, 40 mM Tris-HCl, and 1 M NaCl) and LiCl buffers (0.5 M LiCl, 2% NP-40, 1% Na deoxycholate, 2 mM EDTA, and 20 mM and Tris-HCl). The eluted DNA was next purified by phenol-chloroform. NEBNext DNA library prep master mix set for Illumina (E6040L) was used follow the companies instructions.
GEO Accession
GSM3071209