GSM8863079: HEK293T, G3BP1-Lantern, 60min stress, Input, rep1; Homo sapiens; RNA-Seq
For the profiling of subcellular transcriptomes, cells were lyzed with TRIzol. Total RNAs were extracted, clicked with biotin handles, enriched with streptavidin-coated beads, and purified again with TRIzol. For the study of cancer cell migration, cells with strong biotin signals were sorted by flow cytometry and lyzed with 0.5% (v/v) Triton X-100 and 0.1 U DNase I. For the profiling of subcellular transcriptomes, 100 ng Input RNA samples or 5 μL Enrich RNA samples were utilized for NGS library construction. For identifying G3BP1-proximal and OMM-proximal RNAs in the HEK293T cells, rRNAs were removed with Ribo-off rRNA Depletion Kit (Vazyme) following the manufacturer's protocol. The purified RNAs were dissolved in 5 μL nuclease-free water. NEBNext Ultra II RNA Library Prep Kit for Illumina was used for NGS library construction. RNAs were fragmentated to ~300 nt before first strand synthesis. For the study of cancer cell migration, the sorted cells were used for pseudo-bulk RNA-sequencing library construction following the previously published Sherry2 protocol. Libraries constructed from polyA RNAs (OMM-proximal and G3BP1-proximal RNAs of HEK293T cells, and mRNAs of MC38 cells) were sequenced to a depth of 3 G raw base. And libraries constructed from total RNAs (ERM-proximal RNAs of HEK293T cells and G3BP1-proximal RNAs of rat cortical neurons) were sequenced to a depth of 15 G raw base.