Biotin labeled RNAs were synthesized by Scientific TranscriptAid T7 High Yield Transcription Kit (Thermo). PCR primers with T7 promoters were used to amplify DNA templates for RNA synthesis, RNA transcribed in vitro with biotin RNA labelling mix and T7 RNA polymerase, treated with RNase-free DNase I (Roche), and purified with the RNeasy Mini Kit (Qiagen). Cells lysates or purified proteins were incubated with biotin-labeled RNAs overnight. The proteins associated with biotin-labeled RNAs were then pulled down with Streptavidin Magnetic Beads (Thermo) after 1-hour incubation. The proteins was then washed and used for Western blot analysis.
Scientific transcriptaid t7 high yield transcription kit
Manufactured by Thermo Fisher Scientific
The Scientific TranscriptAid T7 High Yield Transcription Kit is a reagent kit designed for the in vitro transcription of RNA from DNA templates using the T7 RNA polymerase. The kit provides the necessary components for efficient and high-yield transcription reactions.
Automatically generated - may contain errors
4 protocols using scientific transcriptaid t7 high yield transcription kit
1
RNA-Protein Interaction Identification
2
RNA-Protein Interaction Identification
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Biotin labeled RNAs were synthesized by Scientific TranscriptAid T7 High Yield Transcription Kit (Thermo). PCR primers with T7 promoters were used to amplify DNA templates for RNA synthesis, RNA transcribed in vitro with biotin RNA labelling mix and T7 RNA polymerase, treated with RNase-free DNase I (Roche), and purified with the RNeasy Mini Kit (Qiagen). Cells lysates or purified proteins were incubated with biotin-labeled RNAs overnight. The proteins associated with biotin-labeled RNAs were then pulled down with Streptavidin Magnetic Beads (Thermo) after 1-hour incubation. The proteins was then washed and used for Western blot analysis.
3
Identifying FILNC1-Binding Proteins
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Biotin-labeled RNAs were synthesized by Scientific TranscriptAid T7 High Yield Transcription Kit (Thermo). Cells were lysed, and incubated with biotin-labeled RNAs overnight. The proteins associated with biotin-labeled RNAs were then pulled down with Streptavidin Magnetic Beads (Thermo) after 1 h incubation. The proteins were then washed and used for Western blotting or mass spectrometry (MS) analysis. Cells used in MS analysis were cultured under 1 mM glucose medium for 24 h, and MS analysis included biotinylated FILNC1 pulldown group, as well as antisense (AS) FILNC1 and streptavidin beads only pulldown groups as negative controls. In the subsequent computational analysis to enrich true FILNC1-binding proteins, we filtered out all the proteins in FILNC1 pulldown group with less than three spectral count, and set up a cutoff for at least three fold peptide enrichment of FILNC1 group as compared to either AS FILNC1 or beads only group. Such analysis generated a list of totally 88 potential binding proteins of FILNC1, including AUF1 (Supplementary Data 1 ). The primer sequences used in these assays are described in Supplemental Experimental Procedures.
4
Biotin-Labeled RNA Pulldown Assay
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Biotin-labeled RNAs were synthesized by Scientific Tran-scriptAid T7 High Yield Transcription Kit (Thermo Fisher Scientific). Briefly, LINC01503 was cloned into pGEM-T vector with T7 promoter (Promega), and then used to amplify DNA templates for RNA synthesis. RNA was transcribed in vitro with T7 RNA polymerase and biotin labeling mix, treated with RNase-free DNase I, and purified with the RNeasy Mini Kit (Qiagen, Hilden, Germany). Cell lysates were incubated with biotin-labeled RNAs overnight. Proteins associated with biotinlabeled RNAs were immunoprecipitated with streptavidin magnetic beads (Thermo Fisher Scientific), and subjected to mass spectrometric analysis or Western blotting analysis.
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