RNA pulldown assays were carried out as described briefly: in vitro biotin-labelled RNAs (SH3PXD2A-AS1, its antisense RNA) were transcribed with Biotin RNA Labeling Mix (Promega Corporation, USA) and T7 RNA polymerase (Thermo Fisher Scientific, USA) treated with RNase inhibitor and purified with a Clean-up kit (Promega Corporation, USA). The biotinylated SH3PXD2A-AS1 probes were dissolved in binding and washing buffer and incubated with streptavidin agarose resin (Thermo Fisher Scientific, USA). Then, H292 cell lysates were incubated with probe-coated streptavidin beads, and the pulled-down proteins were run on SDS-PAGE gels. Then, the gels were stained with Coomassie Blue, and differentially abundant bands were cut out for mass spectrometry (Shanghai Applied Protein Technology Co., Ltd., China).
Clean up kit
Manufactured by Promega
Sourced in United States
The Clean-up kit is a laboratory product designed to purify and concentrate DNA or RNA samples. It removes contaminants and inhibitors, allowing for improved downstream processing and analysis.
Automatically generated - may contain errors
Lab products found in correlation
Streptavidin agarose resin, by Thermo Fisher Scientific (3 mentions)
Biotin rna labeling mix, by Promega (3 mentions)
T7 rna polymerase, by Thermo Fisher Scientific (2 mentions)
Qubit 4, by Thermo Fisher Scientific (1 mentions)
9700 thermocycler, by Thermo Fisher Scientific (1 mentions)
Bioanalyzer 2100, by Promega (1 mentions)
Biotin rna labeling mix, by Roche (1 mentions)
6 protocols using clean up kit
1
Identifying Proteins Interacting with lncRNA
2
Biotin-Labeled RNA Pulldown Assay
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RNA pulldown assays were performed as previously described.21 Briefly, biotin‐labelled RNAs (antisense RNA) were transcribed using Biotin RNA Labeling Mix (Promega Corporation) and T7 RNA polymerase treated with RNase inhibitor and purified with a Clean‐up kit (Promega Corporation). The biotinylated lnc‐CYB561‐5 probes were dissolved in binding and washing buffer and incubated with streptavidin agarose resin (Thermo Fisher Scientific Inc.). Then, H1299 cell lysates were incubated with probe‐coated streptavidin beads, and the pulled‐down proteins were run on SDS‐PAGE gels. Samples were prepared for Western blotting analysis, as described above.
3
Identification of LINC00460 Interactome
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RNA pull-down assay was carried out as described briefly: in vitro biotin-labeled RNAs (LINC00460 and the antisense RNA) were transcribed with Biotin RNA Labeling Mix (Promega Corporation, US) and T7 RNA polymerase (Thermo Fisher Scientific, US) treated with RNase inhibitor, and purified with Clean-up kit (Promega Corporation, US). The biotinylated LINC00460 probes were dissolved in binding and washing buffer and incubated with Streptavidin Agarose Resin (Thermo Fisher Scientific, US). Then cell lysates of HCT116 and SW480 were incubated with probes-coated streptavidin beads, and pull-down proteins were run on SDS-PAGE gels, and then gels were stained by Coomassie Blue staining, and differential bands were cut off for Mass spectrometry (Shanghai Applied Protein Technology Co., Ltd. China).
4
Genome Sequencing of Cupriavidus amalonaticus
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The initial identity of the culture was obtained by carrying out 16S rRNA Sanger sequencing, using 27F and 1492R primers (Frank et al., 2008 (link)) and, for the V4 region, 515F (GTGCCAGCMGCCGCGGTAA) and 806R (GGACTACHVGGGTWTCTAAT) were used. The 16S rRNA amplicon was purified using a Promega clean up kit and the sequenced at the Center of Bioinformatic and Functional Genomics (CBFG) at Miami University. Genomic DNA was isolated using a protocol provided by the JGI for Bacterial genomic DNA isolation using CTAB (William et al., 2004 ). The gDNA was quantitated using Qubit 4.0 and the Qubit dsDNA high sensitivity quantitation kit from Thermo Fisher Scientific. The gDNA was then sent to the University of Delaware sequencing and genotyping center to sequence the whole genome using PacBio RS II technology. The library preparation, sequencing, assembly, and annotation was carried out by University of Delaware sequencing center. The whole genome of C. amalonaticus CJ25 was deposited to NCBI and JGI and is publicly available. NCBI (Assembly GCA_014859035.1 and WGS JADAQU000000000) and JGI/IMG (Study ID: Gs0145800 Project ID: Gp0489996 Analysis ID: Ga0439030 ).
5
Microdroplet PCR Amplicon Library Generation
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A 500 primer pair microdroplet library was synthesized by RainDance Inc. The primer library and a mix that included the template DNA and all the components of the PCR reaction excluding the primers were loaded separately on a RainDance RDT 1000 instrument for merging. In preliminary experiments, we found that as little as 0.5 ng purified gDNA template would produce up to 250 ng post-amplification DNA. For the work described in this manuscript, we used 10 or 20 ng of input DNA. The merged droplets were then amplified using an Applied Biosystems 9700 thermocycler with the following conditions: 94°C for 2 minutes, 55 cycles at 94°C for 15 seconds, 54°C for 15 seconds and 68°C for 10 minutes, with a hold at 4°C. After amplification, the PCR droplet emulsion was broken to release the DNA amplicons. The amplicon was then purified using a Promega clean up kit and quantified using the 2100 Bioanalyzer. The bioanalyzer trace was also inspected to verify that the expected amplicon peak was in the 1000–1100 nt range.
6
Biotin-labeled LINC01594 pull-down assay
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The RNA pull-down assay was carried out as follows. In brief, in vitro biotin-labeled LINC01594 was transcribed with Biotin RNA Labeling Mix (Roche Corporation, US) and T7 RNA polymerase (APExBIO, US), treated with RNase inhibitor, and purified with a clean-up kit (Promega Corporation, USA). The biotinylated LINC01594 probes were dissolved in binding and washing buffer and incubated with streptavidin agarose resin (Beyotime Biotechnology, China). Lysates of HCT116, SW480 and LoVo cells were incubated with probe-coated streptavidin beads, and products were separated by SDS‒PAGE.
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