Co-cultures of colon cancer cells and cancer-associated fibroblasts recapitulate the aggressive features of mesenchymal-like colon cancer

Co-cultures of colon cancer cells and cancer-associated fibroblasts recapitulate the aggressive features of mesenchymal-like colon cancer In order to sort single cells from the co- and mono-culture conditions, single cell suspensions were made. In brief, on day 8 of the experiment droplets were scooped out of the well and resuspended in Liberase to dissolve the co-culture matrix. Cell clusters and organoids were digested into single cells through incubation and resuspension with TrypLE. Viable single cells were sorted in 384-well cell capture plates using the BD FACSAria II. Plates were shipped on dry ice to Single Cell Discoveries, where single-cell RNA sequencing was performed according to an adapted version of the SORT-seq protocol. Cells were heat-lysed at 65° C followed by cDNA synthesis. After second-strand cDNA synthesis, all the barcoded material from one plate was pooled into one library and amplified using in vitro transcription (IVT). Following amplification, library preparation was done following the CEL-Seq2 protocol to prepare a cDNA library for sequencing using TruSeq small RNA primers (Illumina). The DNA library was paired-end sequenced on an Illumina Nextseq™ 500, high output, with a 1x75 bp Illumina kit.