GSM3268041: Input DNA control for SOM-H3K27ac ChIP; Caenorhabditis elegans; ChIP-Seq
Growth:  glp-1(q224) animals were cultured to starvation on NGM plates at 15°C. L1 worms were floated and plated on one 15-cm NGM plate. Worms were cultured at 15°C for four days until gravid. Adult worms were bleached and embryos were incubated with shaking at 15°C for  36-42 hours. L1s were plated to three peptone enriched plates with 50K L1s per plate and cultured at 25°C for 46-48 hours until adult stage. Adult glp-1(q224) animals were harvested by 3X washes with M9. Extraction:  Worms were crosslinked in 50 mL 2% formaldehyde for 30 minutes in a 50 mL conical tube at room temperature. Formaldehyde was quenched by 1 M Tris (pH 7.5) wash. Worms were then washed 2 more times with M9. Worms were transferred to a 15 mL conical tube and washed with 15 mL prechilled FA buffer. Worm pellets were frozen in liquid nitrogen and stored at -80°C. Worm pellets were thawed on ice and 750 µl of FA buffer was added to each sample. Samples were transferred to a 2 mL Kontes Dounce. Samples were Dounced 15 times with the small “A” pestle for two cycles and 15 times with the large “B” pestle for four cycles. Samples were sonicated with a SFX250 sonifier in an ice bath at 22% amplitude with 10 sec on/1 min off pulses for 34 cycles. 100-650 bp DNA fragments were enriched after sonication. 4.4 mg protein was used for each ChIP sample. 5% of lysate was removed for input sample and stocked at -20°C overnight. 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 “A” 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. Library strategy:  ChIP-seq
GEO Accession
GSM3268041