GSM7889837: Pancreas, Healthy, CD45-, sample4; Mus musculus; RNA-Seq
The pancreatic tail was digested in a two-step enzymatic process. First, the pancreatic tail was digested with RPMI 1640 (Nacalai Tesque) containing 2% FBS, 1mg/mL collagenase type XI (Sigma-Aldrich) and 10U/ml DNase I (TaKaRa) for 10 minutes at 37℃, and then with Accumax (Nacalai Tesque) for 5 minutes at 37℃. The single-cell suspension was obtained by passing through a 70 µm cell strainer. The resulting cell suspensions were washed with MEM α (Thermo Fisher Scientific) containing 2% FBS. RNA-seq library was constructed using a modified sciRNA-seq protocol. The CD45 negative cells were sorted into 384-well plates with barcoded oligo dT primer for each well (1 cell / well). After reverse transcription in each well, cDNA was pre-amplified using the xGen™ ssDNA & Low-Input DNA Prep (IDT). Purified cDNAs were tagmentationed with Amplicon Tagment enzyme (Illumina) and PCR for library construction was done by using NEBNext High-Fidelity 2× PCR Master Mix (NEB).