GSM7500356: mCherry_negative_tumor_WTA; Mus musculus; RNA-Seq
Tumors and 1-2 mm of surrounding tissue from DMBA/TPA treated mice were excised and finely minced with a scalpel. Tumor pieces were submerged in 5 mL prewarmed DMEM with 0.25% Trypsin/EDTA and 3 U/mL DNAse I at 37°C on an orbital shaker with rigorous resuspension every 5 minutes for a total of 30 minutes. Reaction was quenched with 1 ml FBS and strained through a 70 µM mesh, pelleted at 1400rpm for 10 minutes with excess PBS and resuspended in 0.5 mL FACS buffer before mCherry-negative sorting. Single-Cell capture was performed with BD Rhapsody using Rhapsody Enhanced Cartridge Kit (BD Biosciences 664887). Reverse transcription from beads and sequencing library production was carried out according to manufacturer's instructions (BD Biosciences, Doc ID: 210967 Rev. 1.0). Whole Transcriptome Amplification Analysis (WTAs) from BD Rhapsody Cartridges were sequenced in full SP flowcells at 1.8nM concentration with 10% PhiX, dual indexing i5 - 8bp, i7 - 8bp and Read1 64 cycles.