GSM6622742: DU102 treated, replicate 1, scRNAseq; Mus musculus; RNA-Seq
Freshly collected liver tumors from MYC-ON mice were dissected by tissue dissection kit (stemcell). The dissected tissues were filted by 40um filter and fellowed by red cell lysis buffer. Then the cells were centrifuged and washed with PBS buffer. Cells were further stained with PE labelled anti-CD45 antibody (1:500 biolegend) and zombie NIR(biolegend), washed and resuspended in FACS buffer (HBSSwo, 2% v/v FCS, 5mM EDTA). Lived tumor infiltrated CD45+ cells were sorted using a FACS Jazz (Becton Dickinson) according to the presence or absence of PE signal and APC-cy7 signal. Library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, sorted CD45+ cells were resuspended in the master mix and loaded together with partitioning 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 Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added.