GSM9512773: hCD19-PDAC cells with Slc33a1 knock-out isolated from tumor, Biological replicate 3; Mus musculus; RNA-Seq
C57BL/6 mice were orthotopically injected with 6x105 hCD19+ PDAC cells with a control (Olfr402) KO, Slc33a1 KO or Keap1 KO. Tumors were isolated after 12 days and dissociated into single cell suspensions. For this purpose, tumors were minced with a scalpel and incubated for 30 min in a HBSS-based digestion buffer containing 10 mM HEPES (Invitrogen, #15630080), 12.5U Collagenase IV (Millipore Sigma, C2674), and 200U DNaseI (NEB, #M0303) at 37°C. Next, tumors were further dissociated using a gentleMACS tissue dissociator (Miltenyi Biotec). Enzyme-activity was quenched with FBS and the samples were filtered through sequential filtering steps to obtain single cell solutions. Single cell solutions were sorted for E2-Crimson+ tumor cells. At least 100,000 tumor cells per sample were submitted in DNA/RNA shield solution (Zymo Research, #R1100) to Plasmidsaurus for bulk RNA transcriptome profiling. Library construction was performed by Plasmidsaurus. For sequencing library preparation, mRNA was converted into complementary DNA (cDNA) via reverse transcription and second-strand synthesis, followed by tagmentation, library indexing, and amplification. Their approach uses 3' end counting to capture differential gene expression.