GSM5827382: KHW_PBMC; Homo sapiens; RNA-Seq
CSF was quickly transported and centrifuged at 700g for 10 min. After centrifugation, the supernatant was removed, and then the pellet was resuspended with 30 uL of CSF. A total of 10 μL of the single-cell suspension was counted in a Cellometer counting chamber (PD100, Nexcelom, MA, USA) through staining with acridine orange and propidium iodide (AO/PI) reagent (CS2-0106, Nexcelom, MA, USA). Only samples having more than 2,000 cells were selected for scRNA-seq. PBMCs were isolated using Lymphoprep (07861, STEMCELL Technologies, BC, Canada) and SepMate-50 tubes (ST86450, STEMCELL Technologies, BC, Canada) according to the manufacturer's protocol. Briefly, the enriched PBMCs were obtained from density gradient–medium centrifugation. The red blood cells were removed with an ACK lysing buffer (A1049201, Gibco, MA, USA). Cells were resuspended in complete RPMI medium containing 10% fetal bovine serum, 1% GlutaMax, and 1% penicillin/streptomycin up to 10 mL. All CSF/PBMC samples were processed without freezing in the RNA workflow. Single-cell cDNA synthesis, amplification, and sequencing libraries were generated using the Single Cell 3′ Reagent Kit (10X Genomics) following the manufacturer's instructions. Sequencing was carried out on an Illumina HiSeqXten and sequenced using the following parameters: Read1: 28 cycles, i7: 8 cycles, i5: 0 cycles, Read2: 91 cycles to generate paired-end reads.