GSM8187561: Multiple myeloma BM sample, melflufen intermediate sensitivity, rep  1; Homo sapiens; RNA-Seq
Viably frozen myeloma patient bone marrow mononuclear cells were thawed in +37°C water bath. Cells were washed with 25% conditioned medium (CM; RPMI 1640 medium supplemented with 10% FBS, 2 mM L-glutamine, penicillin (100 U/ml), streptomycin (100 μg/ml) and 25% conditioned medium from the HS-5 human bone marrow stromal cell line), centrifuged 5 min at 300g RT and supernatant discarded. Cells were resuspended in 25% CM and filtered through a cell strainer to remove any remaining cell debris and large clumps. Cell viability was masured by staining the cells with Trypan blue dye and Countess II cell counter. Cells were centrifuged 5 min at 300g RT and supernatant discarded. These cell were placed in the density of 1M cells / ml PBS with 0.04% BSA. The cells were stained with CD138-APC antibody (clone MI15, BD Biosciences) and 7-AAD viability dye (BD Biosciences) and incubated for 15 min in the dark at RT. Cells were washed with PBS with 0.04% BSA and centrifuged 5 min at 300g RT . Cells were resuspended to plain RPMI at 5 million cells/ml, placed on ice and keptprotected from light. FACS was performed for the  viable CD138 positive (CD138+) and CD138 negative (CD138-) cells using  BD Influx Cell Sorter (BD Biosciences). After CD138+ and  CD138- cells were sorted, the cells were centrifuged 10 min at 300g +4°C and supernatant reuced to 20  µl if there were less than 200,000 CD138+/CD138- cells and to 30 µl  if there were more than 200,000 cells. The CD138+ cells were mixed with CD138- cells in a ratio of 1:1 with a maximum of 800,000 cells per sample. If there were fewer than 400,000 CD138+ cells, then all CD138+ cells were mixed with 400,000 CD138− cells Library preparation was performed on the mixed CD138+ and CD138- cells using the Chromium Single Cell 3′ Gene Expression v3 reagent kit (10x Genomics). The gel beads in emulsion (GEM) generation, cDNA amplification and library preparation were performed according to the manufacturer's instructions.