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| Status |
Public on Jan 23, 2024 |
| Title |
HEK293T, CHIKV-infected, Input, replicate 1 |
| Sample type |
SRA |
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| Source name |
HEK293T
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| Organisms |
Homo sapiens; Chikungunya virus |
| Characteristics |
cell line: HEK293T
cell type: human embryonic kidney
antibody: none
treatment: 12 hours CHIKV infection, MOI 4
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| Treatment protocol |
Stocks of CHIKV strain LR2006-OPY1 were generated in BHK-21 cells and titered by standard plaque assay in HEK293T cells. The viral inoculum was incubated for 1 hr. CHIKV infections of HEK293T cells were carried out at an MOI of 4 for 12 hours.
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| Growth protocol |
HEK293T cells were cultured at 37 ºC and 5 % CO2 in Dulbecco’s modified Eagle’s medium with glutamine (DMEM) (Gibco) supplemented with 10% (v/v) fetal bovine serum (FBS) (Gibco) and 1% (v/v) non-essential amino acids (Gibco).
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Total RNA was extracted using TRIzol (Thermo Fisher) according to the supplier’s protocol. TURBO DNA-free Kit (Thermo Fisher) was used to remove any contaminating DNA from RNA samples. Isolated RNA was further purified by ethanol precipitation and 30-40 µg of total RNA were subjected to poly(A)-selection with Dynabeads Oligo (dT)25 (Thermo Fisher) according to the manufacturer’s instructions. poly(A)+ RNA from CHIKV-infected HEK293T cells (12 hr p.i. MOI of 4) was fragmented for 10 min at 70°C with RNA fragmentation reagent (Thermo Fisher). After fragmentation, the RNA was cleaned up through an ethanol precipitation step. At this stage, a sample of fragmented poly(A)+ RNA was saved as input RNA for later use in cDNA library construction while 5 μg of fragmented poly(A)+ RNA were used per m6A-IP. For each m6A-immunoprecipitation (m6A-IP), 50 μl of slurry of Magna ChIP Protein G magnetic beads (Sigma-Aldrich) were washed twice with IP/wash buffer 20 mM Tris HCl pH 7.4, 150 mM NaCl, and 0.1% NP-40 (v/v). Beads were re-suspended in 100 μl of IP/wash buffer and coated with 5 μg of rabbit anti-m6A antibody (Active motif, 61495) for 45 min at room temperature with rotation. Beads were then washed 3x with IP/wash buffer and m6A-IPs were prepared by mixing the antibody-coated beads with 910 μl of IP/wash buffer, 35 μl of 0.5 M EDTA pH 8.0, 4 μl of murine RNase inhibitor (New England Biolabs), and 40 μg of fragmented total RNA. 1% input samples (10 μl from the total 1 mL m6A-IP mixture) were removed before immunoprecipitation and stored at −80°C. m6A-IPs were incubated overnight at 4°C with rotation. Beads were then washed 3x with IP/wash buffer. IP samples were further incubated with 126 μl of IP/wash buffer, 15 μl of 10% SDS (v/v) and 9 μl of PCR-grade proteinase K (20 mg/mL) (Thermo Fisher) for 30 min at 55°C. After incubation, 150 μl of the supernatant containing the RNA was transferred to a new microcentrifuge tube and 100 μl of IP/wash buffer was added to each sample. RNA was purified with Trizol LS (Thermo Fisher) and ethanol-precipitated together with 1.5 µl of RNA-grade glycogen (Thermo Fisher). Input samples were processed together with m6A-IPs from the proteinase K treatment onwards.
1 to 5 ng of RNA from input and corresponding m6A-IPs were used for NGS library production following the protocol NEBNext Ultra II directional RNA library prep kit for Illumina (New England Biolabs), treating samples as rRNA-depleted and fragmented RNAs.
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
CHIKV-rep1.narrowPeak
CHIKV-rep1-FC.bw
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| Data processing |
Raw reads were adapter trimmed using cutadapt v4.1, which allowed removal of reads with low quality bases (q<20), short lengths after trimming (<25 bp) and orphan read pairs. The resulting fastq files were mapped using STAR v2.7.6a with parameters --outFilterType BySJout against a merger of the hg38 reference genome and the given viral genome, added as an additional contig. m6A peaks were called using m6aViewer v1.6.1 with default parameters and MACS2 using parameters -q 0.01 --nomodel --extsize 100 -B --SPMR --bdg --keep-dup all -f BAMPE. R software was used to filter significant peaks, defined as showing > 2-fold enrichment over input and a false discovery rate (FDR) < 5% across two biological replicates. Conserved viral peaks found by both analyses were considered significant, with the fold-change over input at peak maxima displayed within genome browser visualization using the output from MACS2 bdgcmp –m FE. Motif analysis across conserved m6AViewer-called m6A peaks was performed using HOMER v4.11 with parameters -rna -len 5,6 -size 50, which identified motifs within the flanking 100 bases of sequence surrounding the peak center.
Assembly: hg38, LR2006_OPY1
Supplementary files format and content: bigwig, narrowpeak
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| Submission date |
May 05, 2023 |
| Last update date |
Jan 23, 2024 |
| Contact name |
Juana Diez |
| E-mail(s) |
juana.diez@upf.edu
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| Organization name |
Pompeu Fabra University
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| Street address |
Dr. Aiguader, 88
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| City |
Barcelona |
| ZIP/Postal code |
08003 |
| Country |
Spain |
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| Platform ID |
GPL33379 |
| Series (2) |
| GSE231737 |
N6-methyladenosine modification is not a general trait of viral RNA genomes [CHIKV] |
| GSE231739 |
N6-methyladenosine modification is not a general trait of viral RNA genomes. |
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| Relations |
| BioSample |
SAMN34598791 |
| SRA |
SRX20230533 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
| Processed data provided as supplementary file |
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