GSM2545810: L3 ChiP-seq; Caenorhabditis elegans; ChIP-Seq
Fragmented chromatin was obtained by sonicating animal pellets on ice in 0.7 mL HLB buffer using a Misonix Ultrasonic Liquid Sonicator S-4000 with the following settings; 50% amplitude, 5 second pulses with a 1 minute pulse-off period between each set of pulses. Each immunoprecipitation was performed using 2.2 mg of protein equivalent in the lysate as previously described with either rabbit anti-GFP IgG fraction (Life Technologies) or control normal rabbit IgG (Life Technologies) antibodies. 50 mL of total lysate was used as non-treated input control.  Immunoprecipitated DNA was collected by phenol-chloroform-isoamyl alcohol recovery. DNA from each immunoprecipitation was run on a 1% agarose gel with ethidium bromide staining and fragments of length 100-300 bp were excised and gel purified using the Omega Gel Extraction Kit (Omega BioTek) as per the manufacturer’s protocol. Immunoprecipitated DNA samples along with purified input DNA for each developmental time point were sent to the Genomics Core Facility at Louisiana State University Pennington Biomedical Research Center for quality control analysis and next-generation sequencing using the Life Technologies SOLiD 5500xl system. 5500 SOLiD Fragment Library Core Kit (4464415)