Instrument: Illumina HiSeq 4000
Strategy: ChIP-Seq
Source: GENOMIC
Selection: ChIP
Layout: PAIRED
Construction protocol: Synchronized L4s were washed with M9 buffer and flash-frozen as “worm popcorn” in liquid nitrogen. The popcorn was pulverized using a biopulverizer before further grinding to a fine powder using a mortar. The powder was dissolved in 10 vol 1,1 % formaldehyde in PBS+1 mM PMSF and fixed for 10 min with gentle rocking. Quenching was achieved by adding 2,5 M Glycine to a final concentration of 125 mM and gently rocking for 5 min. After centrifugation the pellet was washed with ice-cold PBS+1 mM PMSF, before it was resuspended in FA-buffer (50 mM HEPES/KOH pH 7,5, 1mM EDTA, 1% Triton X-100, 0,1 % sodium deoxycholate, 150 mM NaCl) + 1 % Sarkosyl + protease inhibitor and sonicated twice using a Bioruptor (15 times, 15 sec ON, 15 sec OFF; high settings) followed by 15 min centrifugation at full speed, 4°C. The supernatant was taken off (approx. 2-4 mg protein) and incubated either with MRG-1 antibody (Novus) or with buffer ON at 4°C on a rotator. Next, samples were incubated with µMACS ProteinA beads (Milteny Biotec) for 1 h on ice before they were applied to µMACS magnetic M columns that were equilibrated using FA buffer. The columns with bound material were washed 2x using FA buffer followed by washing with FA buffer + 1 mM NaCl and FA buffer + 500 mM NaCl. After further washing with TEL buffer (0,25 mM LiCl, 1 % sodium deoxycholate, 1 mM EDTA) and 2x with TE buffer, the samples were eluted using elution buffer (1 % SDS, 250 mM NaCl, 10 mM Tris pH 8,0, 1mM EDTA). The fixation was reverse crosslinked using 2 µl of 10 mg/ml Proteinase K at 50°C for 1 h followed by incubation at 65°C ON. The DNA was purified using the QIAquick PCR purification kit in a final volume of 40 µl. Libraries were prepared using the NEXTflex qCHIP-Seq v2 kit (NOVA-5130-12) according to manufacturer's instructions.