The genetic profiles based single cell level in a murine model of pancreatic carcinogenesis

We tested how elevated METTL3 expression affects pancreatic cancer in a mouse model. We performed single-cell RNA-sequencing of pancreatic cancer tissue from control (EL1) and METTL3-overexpressing (WTg). FVB/N-EL1-Luc/EL1-TAg mice (Common Name, EL1-Luc/EL1-TAg, abbreviated as EL1) were used to transgenic mice experiments. Briefly, for co-injection to make EL1-Luc/EL1-TAg, two cDNA was used: the oncogenic cDNAs were prepared from 2.7-kb SV40 Large T-antigen, whereas the reporter cDNAs were prepared from Firefly Luciferase cDNA pGL3-Basic Vector. Each cDNA was fused with rodent pancreatic Elastase I promoter. For the construct of Mettl3 overexpressed transgenic mice, three 6-week-old female BL/6 mice were bled with two 6-week-old male BL/6 mice, and fertilized eggs were taken to perform cDNA injection in vitro to make transgenic animals. For the cDNA injection, the open-reading-flame of Mettl3 was cloned from C57BL/6J mice by PCR, of which sequence were verified; Mettl3 cDNA was fused with chimeric CAG promoter, a composite of the cytomegalovirus (CMV) enhancer with the chicken beta actin promoter (CBA). The Mettl3 overexpressing mice were crossed with EL1 mice to make the double transgenic mice (WTg). Mice were confirmed as expected genotype, by PCR. The WTg mice formed abdominal tumors, which were apparent after 6 weeks; the luminous imaging followed to administration of vivoGlo Luciferin as 150 microgram/mouse(g) confirmed the presence of tumor(s) in pancreatic region under the IVIS imaging system (SPI), whereas the opened observation of abdomen indicated around 2-cm tumor(s), and tumors were processed for the preparation of single cells. Single cell suspensions from pancreatic tumor tissues were processed with the 10x Genomics Chromium Controller according to the protocol described in the Chromium Single Cell 3' Reagent Kits User Guide Chromium Next GEM Single Cell 3' Kit v3.1 (Cat# PN-1000269), Chromium Next GEM Chip G Single Cell Kit (Cat# PN-1000127), and Dual Index Kit TT Set A (Cat# PN-1000215) were applied during processing.