GSM7989276: MM13 primary  tumor GFP negative M4; Homo sapiens; RNA-Seq
Frozen vital pieces of primary and metastatic melanoma MM13 and MM27 H2B-GFP collected from NSG mice were defrost and mechanically and enzymatically digest with collagenase typeIII in a shaking incubator at 37C for the time necessary to reach single cell suspension. Digestion was stopped with medium supplemented with 10% serum and washed with PBS.  Sorting was performed using fluorescence-associated cell sorters (Aria and Melody, BD Bioscience) to isolate GFP positive and GFP negative populations from primary tumors after DAPI staining for live cells and from mice metastatic organs after labeling with human CD298-PE and DAPI for human melanoma and live cells. Sorted cell populations were resuspended in PBS-0.04%BSA for Chromium Next GEM Single Cell protocol. Library was prepared with   10x Chromium  protocol according manufacturer recpommendation(10X Genomics 3′ v3 Single Cell Gene Expression Solution).Briefly, cell suspension was loaded onto each channels of the 10x Chromium Single Cell Chip tand single cells were then captured in GEMs. Captured polyA-mRNAs were reverse transcribed into cDNAs and amplified to generate amplified full-length cDNA tagged with CB and UMI (PCR1).