GSM8758950: Islet cells from Tet2(flox/flox)Tet3(flox/flox) Pdx1 cre+ mouse; Mus musculus; RNA-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 ℃. After the supernatant was abandoned, it was resuspended with 4 ℃ PBS containing 0.2% BSA, with cell concentration control at approximately 1000 counts/μL. Debris and dead cells were removed. Single-cell RNA-Seq libraries were prepared using SeekOne® MM Single Cell 3' library preparation kit (cat.). Briefly, appropriate cells were loaded into the flow channel of SeekOne® MM chip. Cell Barcoded Magnetic Beads (CBBs) were pipetted into flow channel and also allowed to settle in microwells with the help of a magnetic field. Cells in MM chip were lysed to release RNA which was captured by the CBB in the same microwell. Then all CBBs were collected and reverse transcription were performed at 37℃ for 30 minutes to lable cDNA with cell barcode on the beads. Further Exonuclease I treatment were performed to remove unused primer on CBBs. Subsequently, barcoded cDNA on the CBBs was hybridized with random primer which had reads 2 SeqPrimer sequence on the 5' end and could extend to form the second strand DNA with cell barcode on the 3' end. The resulting second strand DNA were denatured off the CBBs, purified and amplified in PCR reaction. The amplified cDNA product was then added to full length sequencing adapter and sample index by indexed PCR. The indexed sequencing libraries were cleanup with SPRI beads, quantified by quantitative PCR and then sequenced on illumina NovaSeq 6000 with PE150 read length.