N6-methyladenosine (m6A) antibody - RIP-seq Grade (sample size)

Figure 1. RNA immunoprecipitation using the Diagenode antibody directed against m6A
RNA Immunoprecipitation was performed on 40 µg HeLa total RNA spiked with 0.5 µg of an in vitro prepared transcript containing m6A nucleotides, using 2 µg of the Diagenode m6A antibody (Cat. No. C15410208). An equal amount of IgG was used as negative control. The immunoprecipitated RNA was subsequently analyzed by qRT-PCR with primers specific for the transcript and for an intergenic region, used as negative control. Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

Figure 2. RNA immunoprecipitation using the Diagenode antibody directed against m6A
RNA Immunoprecipitation was performed on 40 µg HeLa total RNA fragmented to a mean size of ~500 bases. A titration consisting of 1, 2, 5 and 10 µg of antibody per RIP experiment was analyzed. IgG (2 µg/IP) was used as a negative IP control. Quantitative RT-PCR was performed with primers for 3’ UTR of the C1RL and SLC35E1 genes, and for exon 12 of the AFF1 gene, used as positive controls, and for exon 5 of the EIF2S3 gene, used as negative control.
Figure 1 shows the recovery, expressed as a % of input (the relative amount of immunoprecipitated DNA compared to input DNA after qPCR analysis).

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Figure 3. RIP-seq results obtained with the Diagenode antibody directed against m6A
RIP was performed with 2 µg of the Diagenode antibody against m6A (Cat. No. C15410208). The IP'd DNA was subsequently analysed on an Illumina HiSeq 4000. The 50 bp tags were aligned to the human genome using the BWA algorithm. Figure 3 shows the signal distribution along the complete sequence of the human X-chromosome (figure 3A) and in three genomic regions surrounding the C1RL, SLC35E1 and AFF1 positive control genes (figure 3B, C and D).

Figure 2. Dot blot analysis using the Diagenode antibody directed against m6A
To demonstrate the specificity of the Diagenode antibody against m6A (Cat. No. C15410208), a Dot Blot analysis was performed using synthetic oligonucleotides containing different modified bases. 150 and 50 pmol of the respective oligo’s were spotted on the membrane. The antibody was diluted 1:400 in PBS-T containing 10 % skimmed milk and 1% BSA. Figure 1 shows a high specificity of the antibody for the oligonucleotide with the N6-methyladenosine modification.