GSM3861100: IGN-H3K27ac ChIP rep2; Caenorhabditis elegans; ChIP-Seq
Growth: VC2010 worms were grown to starvation on NGM plates. Starved L1s were floated on peptone-enriched plates, and grown until gravid at 20Ë?C. Gravid adults were bleached and synchronized by L1 starvation.  L1s were plated to enriched plates and grown until the young adult stage. Animals for nuclei isolation were collected from eighteen enriched plates (~1 million) per ChIP-seq experiment. Young adult animals were harvested at 54-56 hours after plating synchronized L1s when most of the animals had 4-10 embryos. Worms from every six plates (~0.3 million) were collected into one 50 mL conical tube and spun at 3100 rpm for 2 minutes and then washed 3x in M9. Extraction/nuclear preparation:  worms were crosslinked in 50 mL 2% formaldehyde for 30 minutes in three 50 mL conical tubes at room temperature. Formaldehyde was quenched by 1 M Tris (pH 7.5) wash. Worms were then washed two more times with M9. Worms were then washed in the same tubes with 10 mL of prechilled Nuclei Purification Buffer. Germ nuclei were isolated following our protocol. Most of the supernatant was removed so that ~20 ÂµL NPB was left. The nuclei were gently pipetted to mix and then flash frozen in liquid nitrogen and stored at -80Â°C until sonication. IGN samples were sonicated at 2Â°C in a water bath sonicator (Misonix S-4000). 20% amplitude and 10 sec on/10 sec off pulses were used for a total processing time of 20 minutes, resulting in enrichment for 100-650 bp DNA fragments. 5% of lysate was removed for the input sample and stored at -20Â°C until the following day to prepare input DNA. 5 Î¼g of anti-H3K27ac was used for each IGN sample. Library construction:  The Yale Center for Genome Analysis (YCGA) prepared the library and performed sequencing. The KAPA Hyper Library Preparation kit was used for ChIP sequencing library prep. DNA fragment ends were repaired with T4 DNA Polymerase, and Polynucleotide Kinase and â??Aâ? base added using Klenow fragment in a single reaction followed by ligation of custom adapters (IDT) using T4 ligase. Adaptor-ligated DNA fragments were purified and size selected with Agencourt AMPure XP magnetic beads.  Adaptor-ligated DNA fragments were amplified by LM-PCR using custom-made primers (IDT). During LM-PCR, unique 10 base indices were inserted at each DNA fragment and amplified products were purified.