rip  (New England Biolabs)

Journal: Cells

Article Title: Modification of Adenosine196 by Mettl3 Methyltransferase in the 5’-External Transcribed Spacer of 47S Pre-rRNA Affects rRNA Maturation

doi: 10.3390/cells9041061

Figure Lengend Snippet: Characterization of RNA methyltransferase Mettl3 interactions with 5′-ETS of 47S pre-rRNA. ( A ) Scheme of the RT-qPCR primer locations on the pre-47S rRNA. ( B ) Relative amounts of rRNA fragments in immunoprecipitates obtained with anti-m6A antibodies from control and Mettl3 KD cells, quantified by RT-qPCR analysis (* p

Article Snippet: Immunoprecipitation (IP) of m6A-modified RNA using an EpiMark® N6-Methyladenosine Enrichment Kit (E1610S, New England Biolabs, Ipswich, MA, USA) was carried out following a manufacturer’s protocol.

Techniques: Quantitative RT-PCR

Journal: Genes

Article Title: N6-Methyladenosine Landscape of Glioma Stem-Like Cells: METTL3 Is Essential for the Expression of Actively Transcribed Genes and Sustenance of the Oncogenic Signaling

doi: 10.3390/genes10020141

Figure Lengend Snippet: Methyltransferase-like 3 (METTL3)-dependent N 6 -methyladenosine (m 6 A) peak distribution and transcriptome regulation in glioma stem-like cells (GSCs). ( A ) A consensus sequence for m 6 A modification was constructed from the Top 1000 m 6 A peaks using MEME ( www.meme-suite.org ). ( B ) m 6 A peak distribution was plotted along a normalized transcript composed of three rescaled segments of 5′UTR, exon and 3′UTR. Peaks were strongly enriched near the stop codon, which extended into the 3′UTR. ( C ) The distribution of m 6 A peaks in non-targeting shRNA (shNT)-GSCs across different RNA functional regions. ( D ) The number of peaks and genes identified in shNT and METTL3 shRNA (shMETTL3) m 6 A RNA immunoprecipitation (RIP) samples. The number of peaks (left panel) and corresponding genes (right panel) which were lost after METTL3silencing are shown. A global inhibition of m 6 A modification was observed in METTL3-silenced cells. ( E ) An integrated analysis representation of differential m 6 A peaks and transcripts post-METTL3-silencing. Genes which were altered post-METTL3-silencing at the RNA level and had lost or reduced peaks in shMETTL3-GSCs were direct targets regulated at the transcript level. Genes that showed differential regulation with METTL3silencing without m 6 A peaks were considered indirect targets. The number of peaks and corresponding number of genes are shown for each group. Direct targets were divided into two groups based on differential RNA regulation. ( F ) Heat map of differentially regulated protein-coding genes (indirect target) between shNT-GSCs and shMETTL3-GSCs in duplicates. Red indicates high and green low expression with the log 2 ratio scale of 1 to −1.

Article Snippet: 2.4. m6 A RNA Immunoprecipitation Enrichment Epimark m6 A enrichment kit (E1610S, NEB) was used according to the manufacturer’s instructions to perform m6 A RIP.

Techniques: Sequencing, Modification, Construct, shRNA, Functional Assay, Immunoprecipitation, Inhibition, Expressing

Journal: PLoS Biology

Article Title: Refined RIP-seq protocol for epitranscriptome analysis with low input materials

doi: 10.1371/journal.pbio.2006092

Figure Lengend Snippet: Low/high salt-washing method outperforms competitive elution method. (A) Schematic diagram of 3 strategies of m 6 A MeRIP. (B) S/N ratio of GLuc/CLuc was highest in Method II using low/high salt washing. An RNA mixture containing equal amounts of the m 6 A modified control RNA GLuc, the unmodified control RNA CLuc, and NEB antibody were used for m6A MeRIP. (C) S/N ratio of GLuc/CLuc was further increased in a second round of IP using Method II. S/N ratio of GLuc/CLuc (panel D) and SETD7 / GAPDH . CLuc, unmodified control RNA; GLuc, m 6 A-modified control RNA; IP, immunoprecipitation; m 6 A, N6-Methyladenosine; m 6 A MeRIP, m 6 A RNA immunoprecipitation followed by high-throughput sequencing; NEB, New England Biolabs; S/N, signal-to-noise.

Article Snippet: We used the following antibodies to m6 A: rabbit polyclonal anti-m6 A (202 003, SySy, Germany; ABE572, Millipore, Germany) and rabbit monoclonal anti-m6 A supplied in EpiMark N6-Methyladenosine Enrichment Kit (E1610S, NEB, Ipswich, MA). pCMV-Flag-MS2-METTL3 full-length and pCMV-Flag-MS2-METTL3 1–200AA plasmids were generously provided by Professor Richard I. Gregory (Harvard University).

Techniques: Modification, Immunoprecipitation, Next-Generation Sequencing

Journal: PLoS Biology

Article Title: Refined RIP-seq protocol for epitranscriptome analysis with low input materials

doi: 10.1371/journal.pbio.2006092

Figure Lengend Snippet: Comparison of 3 different m 6 A antibodies for MeRIP. (A) S/N ratio of GLuc/CLuc and SETD7 / GAPDH with different antibodies. The amount of 32 μg total RNA from human lung cancer cell line A549 with spiked-in control RNA GLuc and CLuc was used for m 6 A MeRIP using Method II. (B) m 6 A peak signals of SETD7 transcripts in 3 MeRIP-seq libraries. (C) Overlap of m 6 A peaks from the SySy, NEB, and Millipore libraries. (D) Number of m 6 A peaks called by subsampling to different read depths with different antibodies. (E) The percentages of m 6 A peaks in 5 nonoverlapping transcript segments: TSS; 5’UTR; CDS; stop codon; and 3’UTR. (F) Metagene profiles depicting sequence coverage in windows surrounding the TSS and stop codon demonstrated that m 6 . CDS, coding sequence; CLuc, unmodified control RNA; GLuc, m 6 A-modified control RNA; m 6 A, N6-Methyladenosine; m 6 A MeRIP, m 6 A RNA immunoprecipitation followed by high-throughput sequencing; NEB, New England Biolabs; S/N, signal-to-noise; SySy, Synaptic Systems; TSS, transcription start site; UTR, untranslated region.

Article Snippet: We used the following antibodies to m6 A: rabbit polyclonal anti-m6 A (202 003, SySy, Germany; ABE572, Millipore, Germany) and rabbit monoclonal anti-m6 A supplied in EpiMark N6-Methyladenosine Enrichment Kit (E1610S, NEB, Ipswich, MA). pCMV-Flag-MS2-METTL3 full-length and pCMV-Flag-MS2-METTL3 1–200AA plasmids were generously provided by Professor Richard I. Gregory (Harvard University).

Techniques: Sequencing, Modification, Immunoprecipitation, Next-Generation Sequencing

Journal: Communications Biology

Article Title: N6-methyladenosine modification of the Aedes aegypti transcriptome and its alteration upon dengue virus infection in Aag2 cell line

doi: 10.1038/s42003-022-03566-8

Figure Lengend Snippet: m 6 A modification of Ae. aegypti transcripts analysed by dot blot and methylated RNA immunoprecipitation and sequencing (MeRIP-Seq). a A diagram showing the MeRIP-Seq data analysis procedure. b Confirmation of RNA N 6 -methyladenosine (m 6 A) methylation in Ae. aegypti . Total RNA from Aag2 and Vero cells were extracted and subjected to a dot blot assay using a specific anti-m 6 A antibody. EGFP transcripts were synthetised in vitro and used as negative control. The input RNAs were directly stained with ethidium bromide. c Normalized density of m 6 A peaks between immunoprecipitated (black lines) and input (red lines) samples following MeRIP-Seq indicating enrichment of m 6 A in the IP samples as part of the quality control of the data. d Pie chart showing distribution of m 6 A peaks in Ae. aegypti transcripts regions. e The consensus m 6 A motif DRA*CH (D = G/A/U, R = G/A, * modified A, H = U/A/C) was enriched in the identified m 6 A peaks.

Article Snippet: Total RNA was fragmented using the RNA Fragmentation Reagent (Thermo Fisher) for 15 min. MeRIP was performed using EpiMark N6 -methyladenosine Enrichment kit (NEB).

Techniques: Modification, Dot Blot, Methylation, Immunoprecipitation, Sequencing, In Vitro, Negative Control, Staining