GSM6759240: shMib2.1, scRNAseq; Mus musculus; RNA-Seq
Pooled fresh tumors were collected from three mice in shCtrl, shMIB2#1, and shMIB2#2 groups. Prepare enzyme mix by adding 2.35 mL of RPMI 1640, 100 µL of Enzyme D, 50 µL of Enzyme R, and 12.5 µL of Enzyme A into a gentleMACS C Tube. Cut the tumor into small pieces of 2–4 mm. Single-cell suspensions were prepared using Tumor Dissociation Kit (130-096-730, Miltenyi Biotec) by Miltenyi Biotec gentleMACS Octo Dissociator (130-096-427, Mitenyi Biotec). Resuspend sample and apply the cell suspension to a MACS SmartStrainer (70 µm) placed on a 15 mL tube. Use Red Blood Cell Lysis Solution (10×) (# 130-094-183) to remove erythrocytes or dead cells. Resuspended into single cells with 0.4% BSA for 10x genomics processing. Library was performed according to the manufacter's instructions (Chromium Single Cell V(D)J Reagent Kits (v1.1 Chemistry), 10x Genomics). Briefly, GEMs are generated by combining barcoded Single Cell 5' Gel Beads, a Master Mix with cells, and Partitioning Oil on Chromium Next GEM Chip G. Immediately following GEM generation, 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. Silane magnetic beads are used to purify the 10x Barcoded first-strand cDNA from the post GEM-RT reaction mixture, which includes leftover biochemical reagents and primers. The final libraries contain the P5 and P7 priming sites used in Illumina sequencing.