title: GSM6586321: para-tumor scRNAseq YP_dCCA_N; Homo sapiens; RNA-Seq
library_construction_protocol: Human bile duct tissue was obtained from patients . Place a sterile RNase-free culture dish containing an appropriate amount of calcium free and magnesium-free 1 × PBS on ice, the tissue was transferred into the culture dish and cut it into 0.5 mm2 pieces, the tissues were washed with 1 × PBS, and remove as many non-purpose tissues as possible such as blood stains and fatty layers.Tissues were dissociated into single cells in dissociation solution (0.35% collagenase IV5,2 mg/ml papain, 120 Units/ml DNase I) in 37 ℃water bath with shaking for 20 min at 100 rpm. Digestion was terminated with 1× PBS containing 10% fetal bovine serum（FBS,V/V）, then pipetting 5-10 times with a Pasteur pipette.The resulting cell suspension was filtered by passing through 70-30um stacked cell strainer and centrifuged at 300g for 5 min at 4 °C. The cell pellet was resuspended in 100ul 1× PBS (0.04% BSA) and added with 1 ml 1× red blood cell lysis buffer（MACS 130-094-183, 10×）and incubated at room temperature or on ice for 2-10 min to lyse remaining red blood cells. After incubation, the suspension was centrifuged at 300g for 5 min at room temperature. The suspension was resuspended in 100 μl Dead Cell Removal MicroBeads(MACS 130-090-101) and remove dead cells using Miltenyi ® Dead Cell Removal Kit (MACS 130-090-101). Then the suspension was resuspended in 1× PBS(0.04% BSA) and centrifuged at 300 g for 3 min at 4 °C（repeat twice）. The cell pellet was resuspended in 50 μl of 1× PBS (0.04% BSA). The overall cell viability was confirmed by trypan blue exclusion , which needed to be above 85%, single cell suspensions were counted using a haemocytometer/ Countess II Automated Cell Counter and concentration adjusted to 700-1200 cells/μl.