GSM6732000: hITM_HTO; Homo sapiens; RNA-Seq
Single cells were dissociated from the hITM as described. The cells of two different models were hashtagged with TotalSeq-A antibodies (Biolegend) following the manufacturer's protocol for TotalSeqTM-A antibodies and cell hashing with 10X Single Cell 3' Reagent kit v3.1 (10x Genomics). Approximately 400.000 cells per sample were resuspended in 100 µl Cell Staining Buffer (Biolegend) and 5 µl Human TruStain FcXTM FcBlocking (Biolegend) reagent were added. For the blocking reaction, the cells were kept at 4°C for 10 minutes. 1 µg of TotalSeqTM-Antibody was added to each sample, followed by 30-minutes incubation at 4°C. Afterwards, cells were washed three times with 1 ml Cell Staining Buffer and spun down for 5 min at 350xg and 4°C. Finally, the cells were resuspended in an appropriate volume of PBS- and passed through a 40 µm cell strainer (FlowmiTM Cell Strainer, Merck). Cells were counted in a Neubauer Hemacytometer (Marienfeld) and concentration was adjusted to 1000 cells/µl with PBS-. The hashtagged cells were pooled equally and ~20.000 cells were loaded in the ChromiumTM Controller. The machine creates Gel Bead-In-Emulsions (GEMs) to separate single cells into a nanoliter compartment together with an individual barcode. Reverse transcription, cDNA amplification and the construction of gene expression libraries was performed using the 10x Single Cell 3' reaction kit v3.1 (10x Genomics). Incubation and amplification steps were carried out using a SimpliAmp Thermal Cycler (ThermoFisher). Library quantification and quality control was observed using a QubitTM 4.0 Fluorometer (ThermoFisher) and a 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent). Sequencing was performed on a NextSeq 500 sequencer (Illumina). Library was performed according to the manufacter's instructions (single cell 3' v2 protocol, 10x Genomics). Briefly, GCs 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.