GSM9263637: Moderate benzene exposure15; Homo sapiens; RNA-Seq
Peripheral blood samples were collected in EDTA or heparin-anticoagulated tubes. A total of 4 mL of blood was transferred to a 15 mL centrifuge tube and diluted 1:1 with Dulbecco's Phosphate-Buffered Saline (DPBS). Two fresh 15 mL centrifuge tubes were prepared with 4 mL of Ficoll-Paque™ PLUS (Cytiva) in each. The diluted blood was carefully layered over the Ficoll, maintaining a clear interface between the layers. Samples were centrifuged at 750 × g for 25 minutes at room temperature, with acceleration and deceleration rates set to 9 and 1, respectively. After centrifugation, the PBMCs appeared as a distinct white interface between the plasma and Ficoll layers. This layer was carefully aspirated into a new 15 mL tube and brought up to 10 mL with DPBS. The cells were washed by centrifugation at 250 × g for 10 minutes at 4°C. If red blood cells (RBCs) were present in the pellet, 1 mL of RBC lysis buffer was added, mixed gently, and incubated for 1 minute. Subsequently, 5 mL of DPBS was added, followed by centrifugation at 350 × g for 5 minutes at 4°C. The supernatant was discarded, and the pellet was resuspended in 5 mL of DPBS and centrifuged again under the same conditions. This washing step was repeated once. PBMC concentration, viability, and clumping rate were assessed using a CountStar cell counter. The final cell suspension was adjusted to 1 × 10⁶ cells/mL in DPBS. Prior to scRNA-seq, cell number and viability were re-evaluated, ensuring a viability rate exceeding 85%. scRNA-seq libraries were prepared according to the manufacturer's instructions for the 10× Genomics Single Cell 3′ Gel Bead and Library Kit (v3 Chemistry) (Pleasanton, CA). The final libraries were pooled and sequenced on the Illumina NovaSeq X Plus system with 100G paired-end sequencing in PE150 mode.