GSM8717595: DMSO,lung, scRNAseq; Mus musculus; RNA-Seq
Arter gavage,   lung  carcinoma  mouse tumors from STK-treated and DMSO-treated mice were dissected. Then the tumor were dissociated according to the manufacter's instruction (Miltenyi Biotec, CAS#130-096-730) and  tumor single cell suspension were acquired. DAPI (Sigma‒Aldrich, Cat# D9542) was used to identify live or dead cells. Cell surface maker CD45  staining was performed by adding appropriately conjugated fluores cent primary antibodies (Biolegend, CAS#147712) to the cells and incubating  at room temperature for 1 hour in the dark. After staining, CD45+Cell are sorted through Fluorescence activated cell sorting(FACS). Library was performed according to the manufacter's instruction  (Chromium_Next_GEM_Single_Cell_5_v2_UserGuide_10Xgenomics_CG000331). Brifely, CD45+immune cells were resuspended in the master mix and loaded together with partition oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM).The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an llumina R1 primer sequence,Uique Molecular Identifier(UMI) and 10X Barcode. The pooled barcoded cDNA was then cleaned up with silane Dynabeads, amplified by PCR and the appropriate sized fragments were selected with SPRlselect reagent for subsequent library contraction. During the library construction llumina R2 primer sequence, paired-end constructs with P5 and P7 sequence and a sample index were added.