title: GSM9267743: Sample12, asymptomatic; Homo sapiens; RNA-Seq
library_construction_protocol: After harvested, CAS plaques and mouse carotid arteries were washed in ice-cold RPMI1640 and dissociated using SeekOne® Multi-tissue dissociation kit 2 from Miltenyi Biotec as instructions. DNase treatment was optional according to the viscosity of the homogenate. Cell count and viability was estimated using fluorescence Cell Analyzer (Countstar® Rigel S2) with AO/PI reagent after removal erythrocytes (Miltenyi 130-094-183) and then debris and dead cells removal was decided to be performed or not (Miltenyi 130-109-398/130-090-101). Finally fresh cells were washed twice in the RPMI1640 and then resuspended at 1×106 cells per ml in 1×PBS and 0.04% bovine serum albuminat. scRNA-seq libraries were prepared using the SeekOne® MM Single Cell 3' library preparation kit. Briefly, appropriate number of cells were loaded into the flow channel of the SeekOne® MM chip that has 170,000 microwells; the cells were allowed to settle in microwells by gravity. After removing the unsettled cells, sufficient cell-barcoded magnetic beads (CBBs) were pipetted into the flow channel and allowed to settle in the microwells under a magnetic field. Next, excess CBBs were rinsed out, and the cells in the MM chip were lysed to release RNA, which was captured by the CBB in the same microwell. Subsequently, all CBBs were collected, and reverse transcription was performed at 37 ℃ for 30 min to label the complementary DNA (cDNA) with cell barcode on the beads. Exonuclease I treatment was performed to remove unused primers from the CBBs. Subsequently, thebarcoded cDNA on the CBBs was hybridized with a random primer containing reads 2 SeqPrimer sequence on the 5' end to generate second strand DNA with the cell barcode on the 3' end. The resulting second-strand DNA was denatured from the CBBs, purified, and amplified using PCR. The amplified cDNA product was cleaned to remove unwanted fragments and added to a full-length sequencing adapter and sample index using indexed PCR. The indexed sequencing libraries were cleaned with solid phase reversible immobilization beads, quantified using quantitative PCR (KAPA Biosystems KK4824), and sequenced on an Illumina NovaSeq 6000 with a PE150 read length or a DNBSEQ-T7 platform with a PE100 read length