GSM7597607: UPN73_R372_fixed 18 months, scRNAseq; Homo sapiens; RNA-Seq
Human Peripheral Blood Mononuclear Cells (PBMCs) used in this study are obtained after consent from AML patients. PBMCs were isolated from peripheral blood after centrifugation to separate them from plasma and other peripheral blood components using ficoll-Hystopaque density gradient centrifugation. After the separation of PBMCs from whole blood, the cells were preserved using : Cryopreservation that consists of freezing the cells at -80°C or in liquid nitrogen at -195.79°C for a long period using freezing medium (90% FBS and 10% DMSO), the second method to preserve the cell is the cell fixation using the pre-chiled methanol before storage at -80°C . . Cryopreserved cells in liquid nitrogen were thawed at 37°C. Cells were rinsed with RPMI 1640 medium to remove the DMSO-containing freezing medium. To enrich the samples with live cells, dead cells were removed using the Dead Cell Removal Kit. Before starting cell labeling, a 50-μL aliquot containing 1,000,000 cells suspended in a Cell Staining Buffer (CSB) with >95% viability was prepared for each sample. The antibodies used for labeling were TotalSeq -A0251(HTO1) and -A0252 (HTO2) antibody-oligo (Biolegend). Next, we proceeded to the labeling step. For this, 1μg HTO is added to each cell suspension. The two labeled samples are then incubated for 30 minutes at 4°C. The cells then undergo 3 successive washes with 3.5 mL of CSB and centrifugation at 400 g for 5 minutes at 5°C. Finally, the cells are resuspended at a concentration of 1.106 cells/mL in PBS. To rehydrate methanol-fixed PBMCs, the cells were thawed from -80°C to 4°C and incubated for 5 minutes. After incubation, the cells were centrifuged at 1000 g for 5 minutes, and the methanol was carefully removed. The resulting cell pellet was then resuspended in a rehydration buffer containing 3x SSC buffer, 0.04% BSA, 1mM DTT, and 0.2 U/µl RNase inhibitor. Finally, the cell concentration was determined by counting the cells using the LUNA FL cell counter. This rehydration process allows for the preparation of viable and well-dispersed PBMCs for downstream analyses. Library was performed according to the manufacter's instructions Chromium Next Gem Single Cell 3' Reagent Kits v3.1 with Feature Barcoding technology for Cell Surface Protein (for the cryoconserved cells), and and the user guide single cell 3' v3 protocol, 10x Genomics (for fixed cells). Briefly, PBMCs labeled with/without HTOs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.