title: GSM7697848: NHR66FLAG_2; Caenorhabditis elegans; ChIP-Seq
library_construction_protocol: Exposed animals were collected and washed with 4℃ M9 and phosphate-buffered saline to remove bacteria. Cross-linking of protein and DNA was performed in 2% formaldehyde for 30 minutes at room temperature. Cross-linking was quenched with 100 mM glycine, animals were washed in M9, resuspended in lysis buffer (50 mM Hepes–KOH pH 7.5, 300 mM NaCl, 1 mM EDTA, 1% (v/v) Triton X-100, 0.1% (w/v) sodium deoxycholate, 0.5% (v/v) N-Lauroylsarcosine, and protease inhibitors) and lysed with a Teflon homogenizer. Lysates were then sonicated using a Bioruptor UCD-200 for 10 cycles (30 s on, 30 s off) to obtain 500-1000 bp DNA fragments. Sonicated lysates (2 mg) were pre-cleared with protein G Dynabeads (Invitrogen) and incubated with 5 μg anti-GFP antibody (Roche) overnight. Immune complexes were collected with protein G Dynabeads, washed, and eluted from beads. Cross-links were reversed at 65℃ overnight and DNA fragments were purified with PCR purification columns (Qiagen). DNA-ends were repaired, 3'-dA overhang, and ligation of methylated sequencing adaptor. Products were PCR amplified and size selected (100-300bp, including adaptor sequence). Library was qualified for sequencing.