GSM9194127: CBTP-NPs treatment; Mus musculus; RNA-Seq
Subcutaneous M109 tumours from PBS-, BTP-NPs-, and CBTP-NPs-treated mice (n=1/group) were enzymatically dissociated into single-cell suspensions, filtered (40 μm), and processed using 10x Genomics® protocols for low-cellularity inputs (<100,000 cells). Viable cells (>85% viability) were loaded onto a Chromium Controller microfluidic chip (7,000 cells/sample) for barcoding using the Chromium Next GEM Single Cell 3' Reagent Kit v3.1. 1.GEM Generation and Barcode Labeling:​​ Using the Chromium Controller system, load the QC-qualified single-cell suspension, Master Mix (Mix), and 3' Gel Beads containing sequence-tagged barcodes into input channels 1-3 of the chip respectively, along with oil droplets. 2.​Running the Chromium Controller System:​​ Through a microfluidic channel network forming a "Single Cross" intersection system, Gel Beads-in-emulsion (GEMs) are ultimately generated. These are single-cell microreactors encapsulated within oil droplets. During GEM formation, cell lysis occurs, releasing cellular mRNA. This mRNA binds to barcoded oligo-dT primers on the Gel Beads. Reverse transcriptase then synthesizes cDNA. 3.​cDNA Amplification:​​ Add Recovery Agent to mix with the GEMs and remove the oil, releasing the cDNA. The cDNA is subsequently amplified, purified, and enriched. The concentration of the amplified cDNA is quantified using Qubit fluorometric assays to meet library construction requirements. 4.​3' Gene Expression Library Construction:​​ QC-qualified cDNA is used directly to construct 3' gene expression libraries. This involves processes including fragmentation, end repair, ligation of the Read 2 sequencing adapter, and sample indexing. The final libraries undergo quantitative QC to obtain ready 3' gene expression libraries. Libraries underwent paired-end sequencing (PE150) on an Illumina NovaSeq X Plus at 50,000 reads/cell depth.