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

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

结肠癌细胞与癌相关成纤维细胞（cancer-associated fibroblasts）的共培养体系重现了间质样结肠癌的侵袭性特征 实验设计概述：为从共培养与单培养体系中分选单细胞，我们制备了单细胞悬液。简言之，实验第8天，从培养孔中吸出培养滴，重悬于Liberase中以溶解共培养体系的基质。通过TrypLE孵育与重悬，将细胞团与类器官消化为单细胞。使用BD FACSAria II流式细胞仪将活单细胞分选至384孔细胞捕获板中。将样品以干冰运输至Single Cell Discoveries实验室，依据改良的SORT-seq实验流程开展单细胞RNA测序。细胞于65℃下热裂解，随后进行cDNA合成。完成第二链cDNA合成后，将一块板上的所有带条形码的材料混合为一个文库，并通过体外转录（in vitro transcription, IVT）进行扩增。扩增完成后，依照CEL-Seq2实验流程完成文库制备，使用TruSeq小RNA引物（Illumina）构建用于测序的cDNA文库。将DNA文库置于Illumina Nextseq™ 500高产出型测序平台上，采用1×75 bp的Illumina测序试剂盒进行双端测序。