GSM5732355: HFD-O304 islet rep1 - H3K27Me3; Mus musculus; ChIP-Seq
Mice were killed by cervical dislocation and the pancreata were perfused with 2.5 ml cold collagenase (without Mg2+/Ca2+, GIBCO #14180). The pancreata were then dissected out and incubated at 37 °C for 10–15 min. Collagenase activity was stopped by adding 25 ml of Hanks' solution (with Mg2+/Ca2+, GIBCO #14060) and vigorous shaking released the islets from the rest of the pancreatic tissue. Islets were rinsed in icecold Hanks' three times and then picked manually under a stereo microscope. For ChIP-seq experiments ~ 600 islets were processed. Islets were fixed 15 min in 1 ml fixing solution (1% formaldehyde, 50 mM HEPES–KOH, 100 mM NaCl, 1 mM ethylenediaminetetraacetic acid (EDTA), 0.5 mM EGTA) at RT with gentle mixing. Crosslinking was stopped by adding 1/20 volume of 2.5 M glycine for 5 min at RT. Islets were then washed 2 × 5 min at 4 °C with cold 1 × PBS containing protease inhibitor cocktail (Roche, #4693132001), snap frozen and stored at − 80 °C until awaiting further processing. Pooled islets were lysed 5–10 min in 130 μl of lysis buffer at 4 °C and sonicated on a Covaris E220 device (PIP: 105, Duty factor: 2, Cycles/burst: 200, Duration: 9 min). ChIP was performed with rabbit anti-H3K27Ac (Abcam, ab4729) and rabbit anti-H3K27Me3 (Upstate #07-449) antibodies. ChIP-seq libraries were prepared with the Rubicon Thruplex DNA-Seq kit following manufacturer's instructions library preparations and sequencing were performed.
GEO Accession
GSM5732355