Frozen starved L1 worms were ground to a powder, which was incubated in 1.5 mM EGS (Pierce 21565) in PBS for 8 minutes, followed by the addition of formaldehyde to a final concentration of 1%, and incubated for a further 8 minutes. The fixation was quenched for 5 minutes by the addition of 0.125 M glycine. Fixed tissue was washed 2X with PBS with protease inhibitors (Roche EDTA-free protease inhibitor cocktail tablets 05056489001) and once in FA buffer (50 mM Hepes pH7.5, 1 mM EDTA, 1% TritonX-100, 0.1% sodium deoxycholate, and 150 mM NaCl) with protease inhibitors (FA+), then resuspended in 1 ml FA+ buffer per 1 ml of ground worm powder. The extract was sonicated to an average size of ~250 base pairs using a Bioruptor Pico (Diagenode), and 10-20 ug of DNA was used per ChIP reaction. ChIP 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.
GEO Accession
GSM4697041