title: GSM8296166: BN2; Homo sapiens; RNA-Seq
library_construction_protocol: Freshly collected tissue samples were immediately washed with RPMI1640 medium (Gibco, Life Technologies) and dissociated using a Multi-tissue dissociation kit 2 (Miltenyi 130-095-929). The addition of DNase was dependent on the viscosity of the homogenate. Subsequently, cell counts and viability were determined using a fluorescence Cell Analyzer (Countstar® Rigel S2) with AO/PI reagent, following the removal of erythrocytes (Miltenyi 130-094-183). The decision to perform debris and dead cell removal was made based on experimental requirements (Miltenyi 130-109-398/130-090-101). Finally, the freshly isolated cells were again washed twice with medium and resuspended at a concentration of 1×106 cells per ml in 1×PBS and 0.04% bovine serum albumin (BSA) (Thermo Fisher Scientific). Single-cell RNA-Seq libraries were prepared using the SeekOne® MM Single Cell 3' library preparation kit (SeekGene Catalog No.). Briefly, an appropriate number of cells were loaded into the flow channel of the SeekOne® MM chip, which contained 170,000 microwells, and allowed to settle in the microwells by gravity. After removing unsettled cells, Cell Barcoded Magnetic Beads (CBBs) was pipetted into the flow channel and also allowed to settle in the microwells with the assistance of a magnetic field. Next, excess CBBs were removed, 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°C for 30 minutes to label the cDNA with the cell barcode on the beads. Exonuclease I treatment was then carried out to remove unused primers on the CBBs. Following that, the barcoded cDNA on the CBBs was hybridized with a random primer that had the 2 SeqPrimer sequence on the 5' end and could extend to form the second strand DNA with the cell barcode on the 3' end. The resulting second strand DNA was denatured off the CBBs, purified, and amplified in a PCR reaction. The amplified cDNA product was subsequently purified to remove unwanted fragments and added to full-length sequencing adapters and sample indices by indexed PCR. The indexed sequencing libraries were cleaned up with SPRI beads, quantified using quantitative PCR (KAPA Biosystems KK4824), and then sequenced on the Illumina NovaSeq 6000 platform with PE150 read length or the DNBSEQ-T7 platform with PE150 read length.