GSM9847237: HEK293T, SSM library, Round1, 15min, Input; Homo sapiens; OTHER
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. For GLORI2.0 library construction, mRNA was isolated from total RNA by the Dynabeads® mRNA Purification Kit (Invitrogen) according to the manufacturer's instructions. These purified mRNA were fragmented in a 20 μL mixture containing 2 μL 10x RNA fragment buffer (NEB, E6150S), incubated for 5 min at 85oC and immediately placed on ice for 2 min. Subsequently, 2 μL 10x stop buffer (NEB, E6150S) was added. The final solution was purified by Zymo RNA Clean & Concentrator column (Zymo, R1016) according to the manufacturer's instructions. The purified fragment mRNA were added into 50 μL of reaction buffer containing 13 μL glyoxal solution (Sigma, 50649), 10 μL DMSO, 10 μL 5 M NaNO2 (Sigma, S2252), 5 μL saturated H3BO3 solution (Sigma, B0394), 4 μL 500 mM MES (pH 6.0, Sigma, 76039) and nuclease-free water, incubated for 10 min at 50 °C and immediately placed on ice for 2 min for stopping the reaction. The mixture was purified by ethanol precipitation. The pellets were dissolved by 50 μL deprotection buffer containing 0.5 M TEAA (pH 8.6) and 47.5% Formamide. The mixture was incubated at 95°C for 10 min without shaking and immediately placed on ice for 2 min. The mixture was purified by ethanol precipitation. The pellets were dissolved by 8.5 μL nuclease-free water. The RNA library was constructed by SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (TaKaRa, 634413) according to the manufacturer's instructions, except for replacing the RT primer with a ternary primer.