GSM8346373: KL2A drug released, rechallenged with RM-042, Rep 5; Mus musculus; RNA-Seq
Harvested tumors were immersed in cold RPMI-1640 and processed into 2-4 mm3 pieces. Tumor pieces were placed in a solution (25 μg/ml Liberase, 30 U/ml Dnase 1, and 0.01% Hyaluronidase in serum-free RPMI-1640 medium), incubated for 45 minutes at 37°C and dissociated in a gentleMACS Octo Dissociator. Suspensions were strained twice using a 70-μm cell strainer. Cold 10% FBS RPMI-1640 was added to solution to halt enzymatic reaction before the suspension was strained through a 70-μm cell strainer twice. Dead cells and debris were removed using a dead cell removal kit (Miltenyi Biotec Inc, #130090101). Cells were resuspended in PBS with 0.04% BSA and underwent viability assessment using Trypan Blue Stain (0.4%) and the Countess™ II FL cell counter. Library preparation was done following the 10 Genomics Single Cell Chromium 5'V2 protocols. Libraries were pooled to achieve a final concentration of 10 nM, verified by qPCR prior to sequencing. Sequencing was done with a NovaSeq6000 sequencer, utilizing the S4 200 cycles flow cell and sequencing with 26 cycles for read1, 10 cycles for i7 index, 10 cycles for i5, and 90 cycles for read 2 through the ATGC core at MD Anderson Cancer Center.