To compare RNA transcriptomes from bulk cell populations, control or N-cad shRNA cells were dissociated using Accutase at room temperature for 10 min and resuspended in HBSS. 500 cells were collected in the center of a 1.5 ml centrifuge tube containing 4.75 μl SMART-seq reaction buffer (Takara) using a BD FACSymphony S6 (BD Bioscience), avoiding cell loss on the tube walls. To compare RNA transcriptomes from leader and follower cells, ∼90 spheroids of PBT-05 cells expressing histone H2B-Dendra2 cells were allowed to migrate for 24 h on laminin and photoconverted as above. After photoconversion, cells were dissociated with Accutase at room temperature, resuspended in HBSS, transferred to ice, and ∼200 leader and follower cells were sorted into SMART-seq reaction buffer as above. Each experiment was performed on four different occasions.
RNA was prepared and cDNA was synthesized with the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara) and run on the Agilent Tapestation to assess the cDNA product. To construct RNA sequencing libraries, we used Illumina’s Nextera XT kit to fragment the cDNA and added barcoded sequencing adapters. Differential gene expression analysis was performed with the DEseq2 (RRID:SCR_015687) for paired sample R package (Love et al., 2014 (link)). Genes with a Benjamini-Hochberg adjusted P value <0.05 were defined as differentially expressed.
RNA was prepared and cDNA was synthesized with the SMART-Seq v4 Ultra Low Input RNA Kit for Sequencing (Takara) and run on the Agilent Tapestation to assess the cDNA product. To construct RNA sequencing libraries, we used Illumina’s Nextera XT kit to fragment the cDNA and added barcoded sequencing adapters. Differential gene expression analysis was performed with the DEseq2 (RRID:SCR_015687) for paired sample R package (Love et al., 2014 (link)). Genes with a Benjamini-Hochberg adjusted P value <0.05 were defined as differentially expressed.