GSM9700881: Stem cells of apical papilla (SCAP) - sample 2; Homo sapiens; RNA-Seq
Collected tissue samples were transported in ice-cold phosphate-buffered saline (PBS, 1×; calcium- and magnesium-free; Thermo Fisher Scientific® Inc. ) supplemented with 1% Primocin® and transferred to the laboratory within 10 minutes. Tissues were minced into 0.5–1 mm fragments and enzymatically digested using 3 mg/mL type I collagenase and 4 mg/mL Dispase II (Sigma-Aldrich, Merck KGaA, Germany) at 37 °C for 15 minutes, with manual agitation every 5 minutes. Samples were subsequently vortexed for 10 seconds, gently triturated using wide-bore pipette tips, and filtered through a 40 µm cell strainer (Thermo Fisher Scientific© Inc). Cell suspensions were centrifuged at 3000 rpm for 5 minutes, and pellets were resuspended in Minimum Essential Medium (MEM; Gibco) supplemented with 20% fetal bovine serum (FBS; Gibco) and 1% Primocin®. Cells were cultured at 37 °C in 5% CO₂ in 25 cm² flasks and expanded for 10 days. All samples were collected at early passages (P2–P3). Prior to single-cell RNA sequencing, cells were washed with PBS and prepared as single-cell suspensions. The six single-cell suspensions (3 DPSCs and 3 SCAPs) were loaded into the 10X Genomics Chromium® controller and libraries were prepared using the Chromium Single Cell 3′ Reagent Kits v3 according to the manufacturer's protocol, including GEM generation, barcoding, cDNA amplification, and library construction. Libraries were sequenced on an Illumina NovaSeq® platform to a depth of approximately 50,000 reads per cell. Raw sequencing reads were processed to remove adaptor contamination and low-quality reads. Clean reads were aligned to the human reference genome (GRCh38-1.2.0), and gene expression quantification was performed using Cell Ranger count.  Since the analyzed cells were expanded under controlled in-vitro culture conditions, uniformly high RNA integrity and low mitochondrial transcript fractions were expected. Quality control metrics were assessed using distributions of nFeature_RNA (number of detected genes per cell), nCount_RNA (UMIs per cell), and percentage mitochondrial RNA. As per protocol, Cells with fewer than 2200 detected genes or more than 9800 genes were excluded to remove poorly captured cells and potential doublets, respectively. Additionally, cells with mitochondrial RNA percentages exceeding 10% were excluded to remove the clearly defined low-quality tail.