GSM7925602: T2,tumor-PTEN; Mus musculus; RNA-Seq
Place a sterile RNase-free culture dish containing an appropriate amount of calciumfree 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. Tissus were dissociated into single cells in dissociation solution in 37 ℃ water bath with shaking for 20 min at 100 rpm. Digestion was terminated with 1× PBS containing 10% fetal bovine serum, 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. Single-cell suspensions were loaded to 10x Chromium to capture 8000 single cell according to the manufacturer' s instructions of 10X Genomics Chromium Single-Cell 3' kit (V3) .The following cDNA amplification and library construction steps were performed according to the standard protocol. Libraries were sequenced on an Illumina NovaSeq 6000 sequencing system (paired-end multiplexing run,150bp) by Shanghai Personal Biotechnology (Shanghai, China)