GSM8654898: BAPN, Bio Replicate 1, scRNAseq; Mus musculus; RNA-Seq
Fresh tumors were isolated in a sterile biosafety cabinet and placed in ice cold HBSS during transit from mouse facility to laboratory. Tumors were rinsed several times in ice cold HBSS and minced with sterile scalpels. Minced tumor was then enzymatically digested using the Human Tumor Dissociation Kit from Milytenyi Biotec (130-095-929). Post-enzymatic digestion, cells were passed through 100µm and 70 µm strainers (Miltenyi Biotec 130-110-916) to remove debris and undigested fragments. Cell suspensions then underwent several brief rounds of washing in 1X PBS with 3mM EDTA and an incubation in TrypLE (ThermoFisher Scientific 12604013) for 10 min to break apart cell clusters. Cells were suspended in PBS without EDTA and diluted to a concentration of 150k cells/ml for encapsulation. (1CellBio) with an in vitro transcription library preparation protocol (Klein et al., 2015). As per Klein et al., the CEL-Seq work flow is summarized: 1) RT, 2) ExoI, 3) SPRI purification (SPRIP), 4) SSS, 5) SPRIP, 6) T7 in vitro transcription linear Amplification, 7) SPRIP, 8) RNA Fragmentation, 9) SPRIP, 10) primer ligation, 11) RT, 12) library enrichment PCR.