anti double stranded rna  (Absolute Biotech Inc)

Journal: Nature Communications

Article Title: N 6 -methyladenosine modification is not a general trait of viral RNA genomes

doi: 10.1038/s41467-024-46278-9

Figure Lengend Snippet: a Schematic diagram of the genomic RNA (gRNA) and subgenomic RNA (sgRNA) generated during CHIKV infection. b LC-MS/MS quantification of m 6 A modification in poly(A) + RNA isolated from mock- or CHIKV-infected HEK293T and Huh7 cells. Cells were infected for 12 h, at a multiplicity of infection (MOI) of 4. ~100 ng of digested ribonucleosides were analyzed for HEK293T samples and ~75 ng per Huh7 samples. The bar chart shows mean values from three biological replicates with the error bars showing standard deviation (SD). n.s. not significant. ** p < 0.01, using an unpaired two-tailed t -test, p value = 0.0005. Source data are provided with this paper. c Depiction of the qPCR amplicons tiled along the CHIKV RNA for m 6 A-IP-qRT-PCR analysis. d Depiction of the negative (−) and positive (+) control qPCR amplicons in the host SLC39A14 transcript for m 6 A-IP-qRT-PCR analysis. e m 6 A-IP-qRT-PCRs performed with total RNA isolated from different CHIKV-infected cell lines. The RNA was fragmented to ~1 kb. 11 primer sets (one for every kb: CHIKV_1 to CHIKV_11) were tiled along the CHIKV RNA. Bars represent the mean ± SD values of 3 m 6 A-IPs from 3 independent infections for HEK293T and Huh7 cell lines, and from 2 independent m 6 A-IPs for the U2OS cell line. All cell lines were infected for 12 h at an MOI of 4. Source data are provided with this paper. f Intracellular SLC39A14 and viral RNA levels were quantified in total RNA samples by qRT-PCR and normalized against the housekeeping gene GAPDH . For each cell line, SLC39A14 RNA levels were set to 1 and viral RNA levels expressed relative to those of SLC39A14 . The bar chart shows mean values of 3 independent infections with the error bars showing SD. All cell lines were infected for 12 h at an MOI of 4. Source data are provided with this paper.

Article Snippet: The following primary antibodies were used diluted in PBS with 1% (w/v) BSA (Sigma-Aldrich, A7906): anti-METTL3 (Abcam, ab195352, clone [EPR18810], 1:1000), anti-METTL14 (Sigma-Aldrich, HPA038002, 1:250), anti-WTAP (Proteintech, 60188-1-Ig, clone: 4A10G9, 1:250), anti-FTO (Abcam, ab126605, clone [EPR6894], 1:1000), anti-YTHDF1 (Proteintech, 17479–1-AP, 1:1000), anti-double-stranded RNA (clone J2) (Nordic-MUbio, 10010200, 1:2500), anti-CHIKV nsP1 (gift from Prof. A.

Techniques: Generated, Infection, Liquid Chromatography with Mass Spectroscopy, Modification, Isolation, Standard Deviation, Two Tailed Test, Quantitative RT-PCR, Positive Control

Journal: Nature Communications

Article Title: N 6 -methyladenosine modification is not a general trait of viral RNA genomes

doi: 10.1038/s41467-024-46278-9

Figure Lengend Snippet: a Most significantly enriched motif identified in conserved m 6 A peaks across cellular poly(A) + RNA in CHIKV-infected cells. HOMER software was used for motif analysis. b Genome browser tracks showing mapped CHIKV reads (strain LR2006-OPY1) for input and m 6 A-IP samples. The reads are scaled to the same read depth against the viral genome using counts per million normalization. Both biological replicates for each condition (input and m 6 A-IP) are displayed within each track. The common m 6 A peak identified by m6aViewer and MACS2 is indicated. The fold change of m 6 A-IP/input, averaged from two replicates is shown above the called m 6 A peak. HEK293T cells were infected for 12 h at an MOI of 4. The 11 qPCR amplicons generated by the 11 primer sets (CHIKV_1 to CHIKV_11) are indicated. The qPCR amplicon spanning the CHIKV m 6 A peak is also shown. c Genome browser tracks showing mapped reads for input and m 6 A-IP samples across the SLC39A14 transcript. The reads are scaled against the merged genome using counts per million (CPM) normalization. The SLC39A14 qPCR amplicons used as negative and positive controls in m 6 A-IP-qRT-PCRs are also indicated. d Violin and box plot of the log2 fold-change (log2FC) distribution of all 23,539 m6aViewer-called cellular peaks in CHIKV-infected HEK293T cells, conserved between two independent replicates. The SLC39A14 and CHIKV peaks are indicated by red dots. The boxplot shows the median (middle line), 25–75th percentile values and 1.5x interquartile range, while the overlaid violin plot shows the full data distribution. e m 6 A-IP-qRT-PCRs performed to validate the putative m 6 A peak identified via m 6 A-Seq. HEK293T cells were infected for 12 h at an MOI of 4. Total RNA was fragmented to 100–200 nt. The bar chart shows mean values from 3 m 6 A-IPs from 3 independent infections with the error bars showing SD. The CHIKV_10 and CHIKV_11 primer sets were used as negative controls amplifying regions without m 6 A enrichment, according to our m 6 A-Seq data. n.s. not significant ( p > 0.05, using the two-tailed t -test). Source data are provided with this paper.

Article Snippet: The following primary antibodies were used diluted in PBS with 1% (w/v) BSA (Sigma-Aldrich, A7906): anti-METTL3 (Abcam, ab195352, clone [EPR18810], 1:1000), anti-METTL14 (Sigma-Aldrich, HPA038002, 1:250), anti-WTAP (Proteintech, 60188-1-Ig, clone: 4A10G9, 1:250), anti-FTO (Abcam, ab126605, clone [EPR6894], 1:1000), anti-YTHDF1 (Proteintech, 17479–1-AP, 1:1000), anti-double-stranded RNA (clone J2) (Nordic-MUbio, 10010200, 1:2500), anti-CHIKV nsP1 (gift from Prof. A.

Techniques: Infection, Software, Generated, Amplification, Two Tailed Test

Journal: Nature Communications

Article Title: N 6 -methyladenosine modification is not a general trait of viral RNA genomes

doi: 10.1038/s41467-024-46278-9

Figure Lengend Snippet: a Schematic diagram of the SELECT technique . We designed DNA oligos that either annealed to the putative modified DRACH (leaving a gap at the modified site) or to an adjacent non-modified adenosine to serve as a control. The abundance of elongated and ligated products is reduced in the presence of m 6 A modification. The DRACH motif is highlighted with a yellow line. b SELECT technique performed with either total RNA or CHIKV in vitro transcribed (IVT) RNA. HEK293T cells were infected for 12 h at an MOI of 4. The threshold cycle difference of amplification (ΔC T ) of total RNA versus IVT ΔC T is shown for each motif (1–8). If total RNA ΔC T = IVT ΔC T , values would align on the solid diagonal line. If there is a difference of −1 C T cycle between the total RNA ΔC T and the IVT ΔC T , which would indicate the motif is m 6 A-modified, values would align on the diagonal discontinuous line. The lower this C T cycle difference between the total RNA ΔC T and the IVT ΔC T , the more m 6 A modification is present in the DRACH motif. All experiments were performed in three technical replicates (separate SELECT reactions). All C T values from SELECT results can be found in Supplementary Fig. and Source Data. Results are representative of two independent infections.

Article Snippet: The following primary antibodies were used diluted in PBS with 1% (w/v) BSA (Sigma-Aldrich, A7906): anti-METTL3 (Abcam, ab195352, clone [EPR18810], 1:1000), anti-METTL14 (Sigma-Aldrich, HPA038002, 1:250), anti-WTAP (Proteintech, 60188-1-Ig, clone: 4A10G9, 1:250), anti-FTO (Abcam, ab126605, clone [EPR6894], 1:1000), anti-YTHDF1 (Proteintech, 17479–1-AP, 1:1000), anti-double-stranded RNA (clone J2) (Nordic-MUbio, 10010200, 1:2500), anti-CHIKV nsP1 (gift from Prof. A.

Techniques: Modification, In Vitro, Infection, Amplification

Journal: Nature Communications

Article Title: N 6 -methyladenosine modification is not a general trait of viral RNA genomes

doi: 10.1038/s41467-024-46278-9

Figure Lengend Snippet: Stable shRNA HEK293T cell lines were infected for 12 h at an MOI of 4. siRNA-treated (siControl and siYTHDF1) HEK293T cells were infected following 48 h of knockdown for 12 h at an MOI of 4. Scr. scramble, h p.i. hours post-infection. a Western blot quantification analyses are representative of 2 independent infections. β-actin is shown as a loading control. β-actin-normalized values from depleted samples, below each band, are shown relative to their controls. Uncropped blots can be found in Source Data. b Intracellular viral RNA levels were quantified by qRT-PCR and normalized against the housekeeping gene GAPDH . gRNA levels in depleted samples are shown relative to their corresponding controls. The bar chart shows mean values from 3 independent infections with the error bars showing SD. Source data are provided with this paper. c Supernatants collected at 12 h p.i. from CHIKV-infected control and knockdown cells were titered by plaque assay in HEK293T cells. The bar chart shows relative mean values from 3 independent replicates with the error bars showing SD. All statistical analyses were performed using a two-tailed t -test. n.s. not significant, ** p < 0.01, p value = 0.0016. Source data are provided with this paper.

Article Snippet: The following primary antibodies were used diluted in PBS with 1% (w/v) BSA (Sigma-Aldrich, A7906): anti-METTL3 (Abcam, ab195352, clone [EPR18810], 1:1000), anti-METTL14 (Sigma-Aldrich, HPA038002, 1:250), anti-WTAP (Proteintech, 60188-1-Ig, clone: 4A10G9, 1:250), anti-FTO (Abcam, ab126605, clone [EPR6894], 1:1000), anti-YTHDF1 (Proteintech, 17479–1-AP, 1:1000), anti-double-stranded RNA (clone J2) (Nordic-MUbio, 10010200, 1:2500), anti-CHIKV nsP1 (gift from Prof. A.

Techniques: shRNA, Infection, Western Blot, Quantitative RT-PCR, Plaque Assay, Two Tailed Test