GSM8510598: BM MNCs, Healthy Donor, HD10-c; Homo sapiens; RNA-Seq
BM MNCs from two CCUS patients and two eHDs were assessed for concentration and viability using a Life Technologies Countess 3 FL cell counter and trypan blue exclusion staining. Suspensions with 70% or higher viability were used for capture. Two to five sample suspensions were captured for each sample, targeting 50,000 cells per sample. Single-cell capture was performed on the Chromium iX using 10x Genomics' Next GEM 3' scRNAseq V3.1 protocol (CG000315_ChromiumNextGEMSingleCell3-GeneExpression, v3.1, DualIndex_RevE), up to and including cDNA amplification and cleanup. The purified, amplified cDNA was assessed for quality and concentration using an Agilent HS DNA Bioanalyzer and the Qubit HS dsDNA Assay (both from Thermo Fisher). Ten nanograms of the resultant barcoded, full-length cDNA transcripts were used as templates for Nanopore full-length cDNA library preparation. The transcripts were first amplified with custom biotinylated primers. The resultant biotinylated cDNA was captured following bead cleanup using streptavidin beads. A second round of PCR was performed on the eluted cDNA, and the product was used to generate nanopore libraries using the Ligament Sequencing Kit V14 (Nanopore, SQK-LSK114). Purified libraries were quantified using the Qubit HS dsDNA Assay, and 100 fmoles of library were loaded to R10.4.1 flow cells (FLO-PRO114M) and sequenced on a PromethION24 sequencer. Libraries were sequenced for 72 hours with a 200-bp minimum read length using the super accurate base-calling mode. Single-cell full-length cDNA libraries were prepared using the 10X Genomics microfluidics-based Chromium platform. Subsequently, these libraries underwent sequencing on a Nanopore PromethION Flow Cell. Each PromethION Flow Cell typically yields approximately 80 million cell-assigned reads, allowing for the analysis of up to around 8,000 cells per flow cell. Multiple libraries were prepared from the same sample to ensure an adequate number of cells and achieve a higher sequencing depth, and several sequencing runs were conducted for each library.