GSM9457108: T2; Mus musculus; RNA-Seq
Mouse colorectal cancer tissues were harvested from tumor-bearing mice. Tissues were collected immediately after euthanasia.Collected tissues were rinsed with PBS to remove blood and debris. They were then cut into small pieces (1-2 mm) using a scalpel.The minced tissues were enzymatically digested using a cocktail of enzymes (e.g., 0.1% collagenase IV and 0.1% DNase I) in a shaking incubator at 37°C for 30-60 minutes, ensuring gentle agitation.The digested tissue was passed through a 70 µm cell strainer to obtain a single-cell suspension. The resulting cells were washed with PBS and Cell viability was assessed using trypan blue exclusion or a flow cytometry-based method to ensure a minimum viability of 90%.Single cells were processed using the 10x Genomics Chromium Single Cell 3' Library Kit, following the manufacturer's instructions to prepare the libraries for sequencing. The obtained cDNA was fragmented, repaired, and dA-tailed in one step using a Fragmentation Mix and an End Prep Enzyme Mix. After double-sided size selection, fragments with a length of approximately 300 bp were recovered and both ends were ligated with adaptors. The ligated fragmented cDNA was then amplified by PCR using P5 and P7 primers (P7 allowing for multiplexing) and were purified with beads. The Purified libraries were multiplexed and loaded onto an Illumina NovaSeq 6000 instrument (Illumina) following the manufacturer's instructions. Sequencing was performed using a 2x150 bp paired-end (PE) configuration, with image analysis and base calling done by the NovaSeq Control Software (NCS) and GAPipeline-1.6 on the NovaSeq instrument.