GSM6304276: Pancreas, 10 days old, rep2; Capra hircus; RNA-Seq
GSM6304276
The pancreas tissues were quickly isolated from goats, divided into three portions on ice and rinsed with 1× PBS. One portion of the tissues from twin female goats (n=2/group) was minced into 2-3 mm pieces and then incubated in a 37 °C water bath with multiple enzymes (1.0 mg/mL collagenase IV, 2.0 mg/mL dispase, and 50 U/mL DNase I) at 100 × rpm for 30 min. It was worth noting that the suspension should be taken and counted to adjust the digestion time during the digestion process. The digestion was stopped by adding 10% FBS, followed by a filtration step through 70-μm and 30-μm Smart Strainer (Miltenyi Biotec, Bergisch Gladbach, Germany). The suspension was transferred to a 15-mL centrifuge tube and centrifuged at 400 × g for 7 min at 12 °C, and then the supernatant was discarded. Dissociated cells were centrifuged at 300 × g for 5 min at 12 °C, washed twice with 1× DPBS, and resuspended in 2 mL of 1640 (Gibco). Isolated single cells were applied to Red Blood Cell Lysis Buffer and the MACS Dead Cell Removal Kit (Miltenyi Biotec, Bergisch Gladbach, Germany) to remove red blood cells, dead cells and cellular debris following the manufacturer's recommendations. After that, the cells were counted using a haemocytometer (Thermo Fisher Scientific) and checked for viability via trypan blue (Thermo Fisher Scientific, Waltham, MA, USA). Finally, all the single-cell suspensions of each sample were free of cell debris and had high viability (over 90%). The appropriate volume was calculated for a target capture of 8000 cells, and then the cells were further diluted to a user guide concentration (700–1200 cells/μL) with 1× PBS with 0.04% BSA for 10X Genomics sequencing. The scRNA-seq libraries were constructed using the 10x Genomics 3' Reagent Kits v3.