GSM7743360: pancreatic β cell, HFDG14.5WT, H3K27ac, biol rep2; Mus musculus; ChIP-Seq
Pancreatic tissue was perfused with 0.5mg/mL collagenase P (Roche, 11213873001) dissolved in 1×HBSS buffer containing 10mM HEPES (pH 7.4), 1mM MgCl2 and 5mM glucose. Digestion was terminated by adding cold PBS. Islets were manually picked up and dissociated into single cell suspension with 0.25% trypsin (Thermo Fisher Scientific, 25200114) and incubated for 3-5min at 37℃. Fetal bovine serum (FBS) was used to terminate the digestion (40% vol/vol). After centrifugation at 1000rpm for 5min at 4℃, the supernatant was removed and the cell pellet was resuspended with HBSS buffer. Cells were directly sorted using a BD Aria SORP flow cytometer. ChIP-seq was performed following the CUT&RUN protocol. Histone modification ChIPs with primary antibodies against H3K27me3 (Millpore, 07-449), H3K4me1 (Millpore, 07-436) and H3K27ac (Active motif, 39133) were performed using 4,000 Ins1-RFP+ cells, while STAT3 and p300 ChIPs with primary antibodies against STAT3 (CST, 12640) and p300 (CST, D2X6N) were performed using 30,000 cells. ChIP DNA libraries were constructed using the KAPA Hyper Prep kit (Roche, kk2602). The miniATAC-seq libraries were prepared using 1,000 Ins1-RFP+ cells. ATAC DNA libraries were constructed using the TruePrep DNA Library Prep Kit V2 for Illumina (Vazyme, TD502).