GSM9154931: BMMCs from MM patient 3, high-dose melphalan following auto-HSCT, TCR, rep3; Homo sapiens; RNA-Seq
Standard density gradient centrifugation was used to isolate Bone Marrow Mononuclear Cells (BMMCs) from fresh blood samples, followed by washing and red blood cell lysis. Cell viability and quality control were assessed using acridine orange/propidium iodide (AO/PI) dual fluorescent staining on the LUNA instrument. The quality control criteria were as follows: cell concentration of 1000–1500 cells/μL, cell viability >90%, aggregation rate <5%, and cell diameter <30 μm. After confirming that the cell concentration and viability met the required standards, the cell suspension was placed on ice and processed for the 10× Genomics single-cell transcriptomics workflow within 30 min. Using single cell 5 ′ Library and Gel Bead Kit V2 (10x Genomics, PN-1000263) and Chromium Single Cell K Chip Kit (10x Genomics, PN-2000182), the cell suspension was loaded onto the Chromium single cell controller (10x Genomics) to generate single-cell gel beads in the emulsion according to the manufacturer's protocol. Captured cells were lysed and the released RNA was barcoded through reverse transcription in individual gel bead-in-emulsions (GEMs). The next steps were carried out according to the manufacturer's user guide (Chromium Single Cell V(D)J Reagent Kits User Guide, including GEM-RT incubation, post GEM-RT Dynabead cleanup, and cDNA amplification. 5' cDNA libraries were then sequenced on an Illumina NovaseqX sequencer.