GSM2333101: HPL-2_YA_rep1; Caenorhabditis elegans; ChIP-Seq
Frozen worm pellets were ground, crosslinked for 10 minutes in 1.5 mM EGS in FA buffer, then formaldehyde added to 1% and cross-linking continued for an additional 10 minutes (protein factors), or fixed 10 minutes in 1% formaldehyde (histone modifications). Fixative was quenched with 125 mM glycine and cross-linked tissue washed 2X with PBS + protease inhibitors, then resuspended in FA buffer and subjected to sonication in a Bioruptor or Bioruptor pico to an average DNA size of 200bp. Extracts were spun down and the soluble fraction used for ChIP. For ChIPs of protein factors, 1mg of ChIP extract was incubated with 5ug antibody; for histone modifications, 500ug ChIP extract was incubated with 2ug of antibody. After overnight incubation with rotation at 4C, 40ul of equilibrated magnetic beads (coupled to protein A or G, depending on antibody) were added and incubated for 2 hrs at room temperature with rotation. Washes with FA, 500mM-salt FA, 1M salt FA, TEL, and TE buffer were performed and DNA was eluted in elution buffer (1% SDS in TE with 250 mM NaCl) two times with 57 ul volume each, at 65°C. Samples were treated with RNase, proteinase K and then crosslinks were reversed overnight at 65°C. DNA was purified on Qiagen PCR purification columns and used for ChIP library preparation. ChIP or input DNA is blunt ended, A-tailed, ligated to adaptors, amplified by PCR, then size selected using AMPure beads. Illumina TruSeq adaptors and barcodes were used. Samples were sequenced on a HiSeq1500.