rip  (New England Biolabs)

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: Nature Communications

Article Title: An epitranscriptomic mechanism underlies selective mRNA translation remodelling in melanoma persister cells

doi: 10.1038/s41467-019-13360-6

Figure Lengend Snippet: N6-methyladenosine (m 6 A) is enriched in the mRNAs translationally upregulated in persister cells. a 5′-UTR minimum free energy and length of the mRNAs translationally upregulated and top 180 downregulated in persister cells. b GC content of the mRNAs translationally upregulated and top 180 downregulated in persister cells. TE: translation efficiency. c The distribution of m 6 A peaks for mRNA upregulated (red) and downregulated (blue) at the translational level in persister cells. The whole population of mRNAs was plotted in green as a control. d Heavy polysome-bound mRNAs were extracted and purified for m 6 A dot plot assay, m 6 A LC/MS-MS assay and m 6 A RNA immunoprecipitation followed by RNA sequencing (m 6 A-seq) in persister vs. parental cells. e Liquid chromatography–mass spectrometry (LC/MS-MS) nucleoside modification analysis. Total RNAs were purified by poly(A) enrichment and were subjected to digestion. Triplicate samples were then subjected to LC/MS-MS quantification. Each type of RNA methylation was first normalized to non-methylated nucleotide base (A or G) and polysome RNA methylation was then normalized to total RNA methylation (input). m 6 A: 6-methyladenosine; m 6 A m : N 6 ,2’- O -dimethyladenosine; A m : 2’- O -methyladenosine; m 1 A: 1-methyladenosine; m 7 G: 7-methylguanosine; A: adenosine; G: guanosine ( n = 3, unpaired t -test, ns: nonsignificant). f Metagene profiles of enrichment of m 6 A modifications across mRNAs corresponding to translationally upregulated transcripts and the top 180 translationally downregulated transcripts in parental and persister cells. Top panel, metagene profiles of m 6 A modifications of total lysate from parental or persister cells; bottom panel: metagene profiles of m 6 A modifications of heavy polysome fractions from parental or persister cells. CDS, coding sequence. g RT-qPCR quantification of m 6 A enrichment in polysome-bound CREBBP mRNA and HPRT mRNA at indicated time points ( n = 3, ** p -value

Article Snippet: m6 A RNA immunoprecipitation Immunoprecipitation of m6 A was adapted from the protocol of EpiMark N6-methyladenosine Enrichment kit (New England Biolabs).

Techniques: Purification, Liquid Chromatography with Mass Spectroscopy, Mass Spectrometry, Immunoprecipitation, RNA Sequencing Assay, Liquid Chromatography, Modification, Methylation, Sequencing, Quantitative RT-PCR

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 (panel E) in 3 replicates of 1 round of IP. Data related to this figure can be found in S1 Data . 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: Antibodies and plasmids 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 A peaks were enriched in the vicinity of the stop codon. (G) Top enriched motifs identified in the SySy, NEB, and Millipore libraries. Data related to this figure can be found in S1 Data . 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: Antibodies and plasmids 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