GSM5203636: H3K9me3.Suv39.DKO.ES.r1; Mus musculus; ChIP-Seq
Followed the Low cell ChIP protocol with minor modifications. Briefly formaldehyde-fixed cells (1% formaldehyde, 10min RT) were lysed in Buffer B (50 mM Tris-HCl, pH 8.0, 10 mM EDTA, 0,5%SDS, 1x protease inhibitors -Roche) and sonicated in a microTUBE on a Covaris S220 until most of the DNA fragments were 200-500 base pairs long (settings: duty cycle 2%, peak incident power 105 Watts, cycles per burst 200). After shearing lysates were centrifuged (10min 4°C 12000g) and supernatant diluted with 1 volume of Dilution Buffer (1mM EGTA 300 mM NaCl, 2% Triton x-100, 0.2% DOC, 1x protease inhibitors-Roche). In parallel, Dynabeads (Protein A or Protein G dependent on the antibody used) were conjugated to the antibody in PCR tubes by rotating 2 hour at 4°C (30rpm). Antibody-conjugated magnetic beads were added to the tube containing the chromatin and incubated 4h at 4°C (30rpm). Beads were washed (20rpm; 10min) four times with 500 ul Buffer A (10 mM Tris-HCl, pH 7.5, 1 mM EDTA, 0.5 mM EGTA,1% Triton X-100, 0.1% SDS, 0.1% Na-deoxycholate, 140 mM NaCl, 1x protease inhibitors) and once with 500 ul Buffer C (10 mM Tris-HCl, pH 8.0, 10 mM EDTA). Beads were then incubated with 70 μl elution buffer (0.5% SDS, 300 mM NaCl, 5 mM EDTA, 10 mM Tris HCl pH 8.0) containing 2 μl of Proteinase K (20mg/ml) for 1 hour at 55°C and 8 hours at 65°C to revert formaldehyde crosslinking, and supernatant was transferred to a new tube. Another 30 μl of elution buffer was added to the beads for 1 minute and eluates were combined and incubated with another 1 μl of Proteinase K for 1 hour at 55°C. Finally, DNA was purified with SPRI AMPure XP beads (sample-to-beads ratio 1:2). NEBNext Ultra II for Illumina, BioLabs E7335S