GSM8758937: Islet cells from Tet2(flox/flox)Tet3(flox/flox) Pdx1 cre+ mouse2; Mus musculus; Bisulfite-Seq
Adult mouse pancreatic islets were isolated by collagenase IV (2 mg/ml; Worthington) digestion, separated by density gradient centrifugation, and handpicked under a stereomicroscope. Islets were cultured in a standard RPMI medium supplemented with 10% FBS. After isolation and extraction, each tube contained 200-300 islets; they were digested with 0.25% Trypsin-EDTA, at 37 ℃ for 2 min. Digestion was stopped using RPMI 1640 containing 10% fetal bovine serum (FBS), blowing evenly. It was centrifuged at 1360 rpm for 5 min at 4 ℃. Genomic DNA was isolated from pancreatic islets by AllPrep DNA/RNA Micro Kit. Briefly, sufficient genomic DNA was digested using the methylation-insensitive restriction enzyme MspI (New England Biolabs, Beverly, MA, USA). The ends of each restriction fragment were filled in and adenosine was added at the 3′-end. Methylated paired-end Illumina adapters were ligated to the ends of the DNA fragments using T4 DNA ligase, and fragments sized 100-200 bp were purified by agarose gel extraction. The purified fragments were treated with sodium bisulfite and then amplified by PCR. The final PCR products were sequenced on HiSeq 2500 (Illumina Inc., San Diego, CA, USA).