The EZ-Magna RNA-Binding Protein Immunoprecipitation Kit (Millipore) was used to perform RIP and MeRIP assays according to the manufacturer’s instructions. The enriched RNAs were detected by qPCR. Primary antibody against NOP58 (NBP1-46846, Novus), IGF2BP1 (63 ab184305, Abcam) or m6A (A-1801, Epigentek, USA) was used in this study. The sequences of primers were separately listed in Additional file 2 : Table S1. These experiments were performed in triplicate.
A 1801
Manufactured by Epigentek
Sourced in United States
The A-1801 is a laboratory instrument designed for the extraction and purification of DNA and RNA from various biological samples. It utilizes a proprietary membrane-based technology to effectively separate and isolate nucleic acids from complex matrices.
Automatically generated - may contain errors
Lab products found in correlation
4 protocols using a 1801
1
RIP and MeRIP Protocol for RNA-Binding Proteins
2
RNA m6A Detection via Crosslinking and Antibody Staining
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Two micrograms of total RNA were deposited on a nylon membrane (FFN10, Beyotime Biotechnology, Shanghai, China), and then the nylon membrane was crosslinked by UV for 3 min. Next, the nylon membrane was stained by using methylene blue. Subsequently, the nylon membrane was blocked for 1 h in blocking buffer, and the membrane was incubated with m6A antibody (1 µg/mL, A-1801, Epigentek Group Inc., Farmingdale, NY) at 4 ℃ overnight. The membrane was incubated with horseradish peroxidase-labeled secondary antibodies at room temperature for 1 h and enhanced chemiluminescent reagent imaging.
3
Analysis of YTHDC1-bound FENDRR and its m6A methylation
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The RNA immunoprecipitation assay was performed by using an RNA Immunoprecipitation (RIP) Kit (Bes5101, BersinBio, Guangzhou, China) following the manufacturer’s instructions. Briefly, 1 × 107 HPAECs were lysed with RIP lysis buffer. After removing DNA, 20 µL of protein A/G bead-conjugated anti-YTHDC1 antibodies (3 µg, 14392-1-AP, Proteintech, IL, USA) and IgG were added to the samples and incubated overnight at 4 °C. After extracting RNA, the expression of FENDRR was detected by qRT-PCR. For m6A-RNA immunoprecipitation (Me-RIP), an anti-m6A antibody (4 µg A-1801, Epigentek Group Inc., Farmingdale, NY) was used. FENDRR extracted from cell lysates was used to measure the m6A-methylated level of FENDRR.
4
RNA Immunoprecipitation of m6A and YTHDC2
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Immunoprecipitation assays of m6A and YTHDC2 were performed with an RNA Immunoprecipitation kit (BersinBio Biotech, China). In short, about 1*107 BMSCs were collected, and the cells were lysed with polysome lysis buffer for 30 min at room temperature. Then, we added DNase to remove DNA impurities, the supernatant was collected after centrifugation at 16100g for 10 min, and then incubated with anti-m6A antibody (1:50, A-1801, EpigenTek, USA) and anti-YTHDC2 primary antibody (1:30, ab220160, Abcam, USA) overnight at 4 °C in a vertical orientation, after which magnetic beads were added and incubated at 4 °C for 1 h to enrich targets. After washing, RNA was extracted, and target gene expression was detected by qRT-PCR.
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