GSM7663116: Pancreas islet, aged, sample1, scRNA-seq; Mus musculus; RNA-Seq
The mice were sacrificed, and the abdominal cavity was opened. And 1-5 ml of cold 0.5 mg/ml collagenase solution was injected into the pancreas from the common bile duct using a syringe. The pancreases were incubated with 5 ml of cold 0.5 mg/ml collagenase solution at 37  C for 3 min. The digested product was filtered through a 0.25 mm nylon filter into a new 50 ml centrifuge tube, and were centrifuged at 200 x g for 1 min, and the supernatant was discarded. Islets were removed with a 200   L pipette and the islets were resuspended in 0.25% trypsin EDTA solution and incubated at 37  C for 5 min. Digestion was stopped by adding 0.4 volumes of cold fetal bovine serum (FBS) and Cells were resuspended in Hanks buffer (pH 7.4) containing 1% FBS. Then, the hypothalamus, pituitary gland, pineal gland, thyroid gland and adrenal gland were subsequently removed from the brain, thoracic cavity or abdominal cavity of each mouse. All tissue except hypothalamus were cut into small pieces using surgical scissors and were incubated with collagenase (0.5% in HBSS) at 37  C for 5-15 min(depends on the type of tissue) until they were dissociated into a single-cell suspension. The digested product was filtered through a 40   m nylon filter into a new 50 ml centrifuge tube, and wished with cold PBS. Then cells were resuspended in Hanks buffer (pH 7.4) containing 1% FBS. Hypothalamus were process into single nuclear suspension by using nuclear separation kit. Library was proformed according to the manufacter's instruction(single cell 3' v3.1 protocol, 10X Genomics). Briefly, prepare single cell master mix, load chromium next GEM chip G, run the chromium controller, transfer GEMs, GEM-RT incubation, post GEM-RT cleanup   Dynabeads, cDNA amplification, cDNA cleanup   SPRIselect and cDNA QC & quantification.