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Anti YTHDF1 and YTHDF2 RIP-seq to identify target mRNAs in mouse cortex and retina

ABSTRACT: To identify the target mRNAs of the m6A reader proteins YTHDF1 and YTHDF2, we carried out anti-YTHDF1 and anti-YTHDF2 RNA Immunoprecipitation (RIP) followed by RNA-sequencing. Using EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore), RNA from E12.5 wild-type mouse cortices and P0 wild-type mouse retinas was pulled down by rabbit polyclonal anti-YTHDF1 (proteintech) and rabbit polyclonal anti-YTHDF2 (proteintech), and then sequenced on Illumina HiSeq3000 platform. The filtered reads were mapped to the mouse reference genome (GRCm38) using STAR v2.5 with default parameters. The resulting bam files were fed to the HTSeq tool to count the number of RNA-seq reads, which was further normalized to calculate FPKM. To determine which gene is enriched, we computed the FPKM from RIP elute to input, and any fold change greater than 2 was considered enriched. From the embryonic cortex, we identified 986 and 1860 mRNAs by anti-YTHDF1 and anti-YTHDF2 RIP-seq, respectively. Anti-YTHDF1 and anti-YTHDF2 RIP-seq in mouse retina identified 2969 and 1638 mRNAs, respectively. This study provides the gene lists which show mRNAs binding with YTHDF1 and YTHDF2 in the mouse cortex and retina.

ORGANISM(S): Mus musculus

PROVIDER: GSE199962 | GEO | 2022/08/05

REPOSITORIES: GEO

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Anti-YTHDF1 and anti-YTHDF2 RIP-Seq to identify the target mRNAs for YTHDF1 and YTHDF2, respectively

Project description:To identify the target mRNAs of the m6A reader protein YTHDF1 and YTHDF2, we carired out anti YTHDF1 and anti YTHDF2 RNA Immunoprecipitation (RIP) followed by RNA-sequencencing. Using EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore), RNA from P6-P8 wild type mouse cerebellum was pulled down by rabbit polyclonal anti-YTHDF1 (proteintech) or polyclonal anti-YTHDF2 (proteintech) and then sequenced on Illumina HiSeq3000 platform. The filtered reads were mapped to the mouse reference genome (GRCm38) using STAR v2.5 with default parameters. The resulting bam files were fed to HTSeq tool to count the number of RNA-seq reads, which was further normalized to calculate FPKM. To determine which gene is enriched, we computed the FPKM from RIP elute to input and any fold change greater than 2 was considered enriched. Finally, Biological replicates of anti-YTHDF1 RIP-Seq and anti-YTHDF2 RIP-Seq identified 506 and 596 mRNAs transcripts, respectively. This study provides gene lists which shows mRNA binding with YTHDF1 and YTHDF2 in mouse cerebellum.

2021-12-10 | GSE153689 | GEO

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Project description:To identify the target mRNAs of the m6A reader protein YTHDF2, we carired out anti YTHDF2 RNA Immunoprecipitation (RIP) followed by RNA-sequencencing. Using EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore), RNA from P0 wild type mouse retinas was pulled down by rabbit polyclonal anti-YTHDF2 (proteintech) and then sequenced on Illumina HiSeq3000 platform. The filtered reads were mapped to the mouse reference genome (GRCm38) using STAR v2.5 with default parameters. The resulting bam files were fed to HTSeq tool to count the number of RNA-seq reads, which was further normalized to calculate FPKM. To determine which gene is enriched, we computed the FPKM from RIP elute to input and any fold change greater than 2 was considered enriched. Finally, Biological replicates of anti-YTHDF2 RIP-Seq identified 1639 transcripts. This study provides a gene list which shows mRNA binding with YTHDF2 in mouse retina.

2022-03-15 | GSE145390 | GEO

Anti-YTHDF2 RIP-Seq to identify YTHDF2 target mRNAs in hippocampus

Project description:To identify the target mRNAs of the m6A reader protein YTHDF2 in mouse hippocampus, we carired out anti YTHDF2 RNA Immunoprecipitation (RIP) followed by RNA-sequencencing. Using EZ-Magna RIP™ RNA-Binding Protein Immunoprecipitation Kit (Millipore), RNA from P40 wild type mouse hippocampus was pulled down by rabbit polyclonal anti-YTHDF2 (proteintech) and then sequenced on Illumina Novaseq 6000. The filtered reads were aligned to the mouse reference genome (GRCm38) using BWA mem (v 0.7.12).Then the MACS2 (version 2.1.0) peak calling software was used to identify regions of IP enrichment over background, followed by the motif detected by Homer (Heinz et al., 2010). Peak related genes are then confirmed by PeakAnnotator. Different peak analysis was based on the fold enrichment of peaks of different experiments. A peak was determined as different peak when the odds ratio between two groups was more than 2. Using the same method, genes associated with different peaks were identified. Finally, Biological replicates of anti-YTHDF2 RIP-Seq identified 408 mRNAs transcripts. This study provides gene lists which shows mRNA binding with YTHDF2 in mouse hippocampus.

2023-01-19 | GSE171791 | GEO

Anti-YTHDF1 and anti-YTHDF2 RIP-Seq to identify the target mRNAs for YTHDF1 and YTHDF2, respectively

Project description:Anti-YTHDF1 and anti-YTHDF2 RIP-Seq to identify the target mRNAs for YTHDF1 and YTHDF2, respectively

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N6-methyladenosine Modulates Messenger RNA Translation Efficiency

Project description:N6-methyladenosine (m6A) is the most abundant internal messenger (mRNA) modification in mammalian mRNA. This modification is reversible and non-stoichiometric, which potentially adds an additional layer of variety and dynamic control of mRNA metabolism. The m6A-modified mRNA can be selectively recognized by the YTH family “reader” proteins. The preferential binding of m6A-containing mRNA by YTHDF2 is known to reduce the stability of the target transcripts; however, the exact effects of m6A on translation has yet to be elucidated. Here we show that another m6A reader protein, YTHDF1, promotes ribosome loading of its target transcripts. YTHDF1 forms a complex with translation initiation factors to elevate the translation efficiency of its bound mRNA. In a unified mechanism of translation control through m6A, the YTHDF2-mediated decay controls the lifetime of target transcripts; whereas, the YTHDF1-based translation promotion increases the translation efficiency to ensure effective protein production from relatively short-lived transcripts that are marked by m6A. PAR-CLIP and RIP was used to identify YTHDF1 binding sites followed by ribosome profling and RNA seq to assess the consequences of YTHDF1 siRNA knock-down

2015-06-09 | E-GEOD-63591 | biostudies-arrayexpress

Anti YTHDF2 RIP-seq to identify YTHDF2 target mRNAs in P0 mouse retinas

Project description:Anti YTHDF2 RIP-seq to identify YTHDF2 target mRNAs in P0 mouse retinas

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Expression data from YTHDF1 or YTHDF2 down-regulated PC9

Project description:The human YTH domain family protein (YTHDF) family is RNA binding protein which specifically recognizes N6-methyladenosine (m6A), and exerts distinct roles in eukaryocytes; YTHDF1 promotes the translation of m6A modified mRNAs collaborating with initiation factors, and YTHDF2 reduces the stability of the m6A-modified transcripts. We used the Nanostring nCounter to detail the differential gene expression by YTHDF1 and YTHDF2.

2021-08-24 | GSE171634 | GEO

Identification of YTHDF1 targeted m6A modified transcripts in lung cancer cells

Project description:In addition to perform the m6A-seq in A549 cells, we sequenced RNA obtained from the immuno-purified complex of YTHDF1 (RIP-seq) to reveal YTHDF1 bound mRNAs, 3,676 genes were shared (m6A-seq+RIP-seq) as high-confident targets of YTHDF1 , which were mapped to cell cycle and tumor (including lung cancer) related signaling pathways in the KEGG pathway database

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Binding in wild type embryos: SoxN.

Project description:Genome-wide binding of SoxN in wild type embryos: SoxNDam (DamID, stage 8-11), SoxND1 (ChIP, stage 8-11), SoxND2 (ChIP, stage 8-11), SoxNPA179 Early (ChIP, stage 7-10), and SoxNPA179 Late (ChIP, stage 11-13). One DamID experiment, four ChIP experiments. Three biological replicates per experiment. Two conditions: Dam (control) vs SoxNDam (sample) for DamID, anti-M-NM-2gal (control, 40-1a antibody from DSHB) vs anti-SoxN (sample, either SoxND1, SoxND2 or SoxNPA179 antibody) for ChIP. The only commercially available antibody is the anti-BetaGal. All the anti-SoxN antibodies are home-made. anti-BetaGal: Developmental Studies Hybridoma Bank (DSHB), 40-1a, mouse, monoclonal (http://dshb.biology.uiowa.edu/beta-galactosidase_2) SoxND1: polyclonal antiserum raised in rabbit immunized with a protein fragment corresponding to amino acids 2-92 of SoxN. Produced by the modENCODE consortium (http://www.modencode.org/). SoxND2: polyclonal antiserum raised in rabbit immunized with a protein fragment corresponding to amino acids 317-417 of SoxN. Produced by the modENCODE consortium (http://www.modencode.org/). SoxNPA179: affinity purified polyclonal antibody raised in rabbit immunized with a small peptide corresponding to amino acids 506-521 of SoxN. Commisioned by our lab and produced by Eurogentec (http://www.eurogentec.com/eu-home.html).

2014-04-23 | E-GEOD-47337 | biostudies-arrayexpress

RNA-sequencing to identify differentially expressed mRNAs after knockout of YTHDF2 in granule cells of mouse hippocampus dentate gyrus

Project description:To identify differentially expressed mRNAs after knockout of YTHDF2 in granule cells of mouse hippocampus dentate gyrus, we perfromed RNA-seq. Total RNA from DG deficient in YTHDF2 and the negative control DG were collected and subjected to construct sequencing libraries using NEBNext® Ultra™ RNA Library Prep Kit for Illumina® (NEB). After sequencencing, the filtered reads were mapped to the mouse reference genome (GRCm38) using HISAT2 v2.0.5 with default parameters. The resulting bam files were fed to featureCounts v1.5.0-p3 to count the number of RNA-seq reads, which was further normalized to calculate FPKM. Differential expression analysis of two groups (cKO vs NC) (three biological replicates per group) was performed using the DESeq2 package (version 1.20.0). Altogether 779 mRNA were differentially regulated, among which 536 mRNA were upregulated after YTHDF2 KD. This study provides a gene list which shows differentially expressed mRNAs after knockout of YTHDF2 in granule cells of mouse hippocampus dentate gyrus.

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