GSM9179375: Elderly7; Homo sapiens; RNA-Seq
Human BM endothelial cells (EC) and mesenchymal stromal cells (MSC) were isolated by mechanical dissociation, enzymatic digestion (0.3% collagenase I, dispase 5 U/ml, 15 min, 37°C), ACK-mediated RBC lysis, and filtration. Cells were stained in PBS + 2% FBS + 2 mM EDTA with lineage (CD3, CD19, CD45, CD64) and population-specific markers, and dead cells were excluded by TO-PRO-3. EC were defined as TO-PRO-3⁻/Lin⁻/CD45⁻/CD235⁻/CD16⁻/CD56⁻/CD9⁺/CD31⁺ and MSC as TO-PRO-3⁻/Lin⁻/CD45⁻/CD235⁻/CD16⁻/CD56⁻/CD31⁻/CD271⁺. Sorting was performed using a BD FACSAria II into PBS + 0.05% UltraPure BSA. Viability was assessed with AO/PI on a Nexcelom Cellometer. scRNA-seq was performed using the Single Cell 3' Reagent Kits v3.1 (10X Genomics) according to the manufacturer's instructions. Approximately 10,000 cells were loaded at a concentration of 1,000 cells/µL on a Chromium Controller instrument (10X Genomics) to generate single-cell gel bead-in-emulsions (GEMs). Each cell was encapsulated with primers containing a fixed Illumina Read one sequence, followed by a cell-identifying 16 bp 10X barcode, a 10 bp Unique Molecular Identifier (UMI), and a poly-dT sequence. A subsequent reverse transcription yielded full-length, barcoded cDNA. This cDNA was then released from the GEMs, PCR-amplified, and purified with magnetic beads (SPRIselect, Beckman Coulter). Enzymatic Fragmentation and Size Selection optimized cDNA size before library construction. Illumina adaptor sequences were added, and the resulting library was amplified via end repair, A-tailing, adaptor ligation, and PCR. Libraries' quality control and quantification were performed using Qubit 3.0 Fluorometer (Life Technologies) and Agilent's 4200 TapeStation System (Agilent), respectively. Sequencing was performed in a NextSeq2000 (Illumina) (Read 1: 28cycles, i7 Index: 10cycles, i5 Index:10 Read 2: 90cycles) at an average depth of 45,000 reads/cell.
GEO
GSM9179375_r1