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| Status |
Public on Aug 24, 2022 |
| Title |
Anti-m6A Wildtype, Sham Exposure, Fetal – 3 |
| Sample type |
SRA |
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| Source name |
Heart
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| Organism |
Mus musculus |
| Characteristics |
tissue: Heart
strain: FVB/NJ
genotype: WT
treatment: Sham Exposure
antibody: anti-m6A polyclonal antibody (item no. ABE572-I; MilliporeSigma, Burlington, MA)
Sex: pooled male and female
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| Treatment protocol |
A high-pressure acoustical generator (HPAG; IEStechno, Morgantown, WV) was utilized for nano-TiO2 aerosolization as has been previously done for rodent inhalation exposure studies. Using a whole-body exposure chamber, a target aerosol mass concentration of 12 mg/m3 of engineered nano-TiO2 was implemented for a period of 360 minutes per day for 6 non-consecutive days, over an 8-day period.
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| Growth protocol |
Friend Virus B NIH Jackson (FVB/NJ) wild type mice (14 females, 7 males at 10 weeks) were purchased from Jackson Laboratory (Bar Harbor, ME). Animals were housed in the West Virginia University Health Sciences Center Animal Facility with access to a standard chow diet and water ad libitum. All animals were allowed a 48-hour acclimation period before being handled. Females were housed in groups of 4 per cage and introduced to male bedding three days prior to being introduced to the males for breeding.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from pooled fetal heart samples (sham, n =3; nano-TiO2, n=3) using QIAzol lysis reagent (item no. 79306, Qiagen). Samples were homogenized in QIAzol using a rotor-stator homogenizer and further processed using the miRNeasy Mini Kit (item no. 217004, Qiagen) per manufacturer’s protocol. Concentrations were determined for each sample using the NanoDrop ND-100 (Thermo Fisher Scientific, Waltham, MA). Spike-in controls obtained from the EpiMark N6-Methyladenosine Enrichment Kit (item no. E1610S; NEB) were prepared following the manufacturer’s protocol. The fetal cardiac total RNA samples (3 µg) were fragmented into ~250-350-nucleotide fragments using the NEBNext Magnesium RNA Fragmentation kit (item no. E6150S; New England Biolabs (NEB), Ipswich, MA) at 65°C for 5 minutes. The Monarch RNA Cleanup Kit (item no T2030L; NEB) was performed per manufacturer’s protocol. Samples were eluted with 20 µL of nuclease-dree water and concentrations were determined using the NanoDrop ND-100 (Thermo Fisher Scientific). 2 µL of fragmented total RNA was saved from each sample to be used as input and the rest was used for m6A RNA immunoprecipitation followed by sequencing (m6A-RIP-seq).
Library preparation was performed on immunoprecipitated (IP) and total (input) RNA samples from sham (n = 3) and nano-TiO2 (n = 3) gestationally-exposed fetal offspring hearts using the NEB® Single Cell/Low Input RNA Library Prep kit for Illumina® (item no. E6420S, NEB) and Illumina® compatible NEB® UDIs (item no. E6440S, NEB), which mitigate sample misassignment due to index-hopped reads. The Agilent 2100 BioAnalyzer (Agilent Technologies, Santa Clara, CA) was employed to determine the size distribution of RNA. Libraries were PCR amplified for 10 cycles and NEB UDIs were used for indexing by amplifying for 8 cycles. The RNA sample concentrations were quantified using a Qubit Fluorometer (Thermo Fisher Scientific). The samples were sequenced using the Illumina NextSeq 2000 (Illumina Inc., San Diego, CA) at Marshall University as paired end (PE) 2x50 bp reads.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
NextSeq 2000 |
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| Description |
The protocol used in our study was adapted from m6A-RIP protocols described in “Refined RIP-seq protocol for epitranscriptome analysis with low input materials” and “Landscape and Regulation of m6A and m6Am Methylome across Human and Mouse Tissues”, with modifications. In a 1.5 mL microcentrifuge tube (per sample), 30 µL of protein-A magnetic beads (item no. 10002D; Thermo Fisher Scientific) and 30 µL of protein-G magnetic beads (item no. 10004D; Thermo Fisher Scientific) were mixed, magnetic field applied, and supernatant removed. The mixture of beads was washed twice with 400 µL of IPP buffer (10 mM Tris-HCl, 150 mM NaCl, pH 7.5; 0.1% IGEPAL CA-630) and resuspended in 500 µL of IPP buffer. 5 µg of affinity purified anti-m6A polyclonal antibody (item no. ABE572-I; MilliporeSigma, Burlington, MA) was added to the beads and incubated overnight at 4°C. An additional tube with the mixture of beads received 5 µg of the normal rabbit IgG control antibody (item no. 2729; Cell Signaling Technology (CST), Danvers, MA) and was also incubated overnight at 4°C. The magnetic field was applied, and supernatant removed, followed by washing the antibody-bead mixture twice with IPP buffer. In a 1.5 mL microcentrifuge tube, a 500 µL mixture was prepared containing 100 µL of 5X IPP buffer, fragmented total RNA (diluted with nuclease-free water to a volume of 395 µL), and 5 µL of RNasin Plus RNase Inhibitor (item no. N2611; Promega, Madison, WI). The antibody-bead mixture was resuspended in the 500 µL mixture containing the fragmented RNA and incubated with orbital rotation at 4°C for 2 hours, followed by application of the magnetic field, removal of the supernatant, and two washes with 1,000 µL of IPP buffer. The bead-antibody-RNA mixture was then washed twice with 1,000 µL of low-salt IPP buffer (10 mM Tris-HCl, 50 mM NaCl, pH 7.5; 0.1% IGEPAL CA-630) and twice with 1,000 µL of high-salt IPP buffer (10 mM Tris-HCl, 500 mM NaCl, pH 7.5; 0.1% IGEPAL CA-630). A competitive elution buffer was then prepared containing 6.7 mM N6-methyladenosine 5’- monophosphate sodium salt (item no. M2780, Sigma-Aldrich) in IPP buffer. Each immunoprecipitation (IP) sample was resuspended in 100 µL of the m6A competitive elution buffer with continuous shaking at 4°C for 1 hour. The magnetic field was applied, which allowed us to collect the supernatant (eluted m6A RNA) into a new tube. The eluted RNA was further purified using the RNeasy MinElute Cleanup Kit (item no. 74204; Qiagen) following manufacturer’s protocol. The m6A enriched RNA was eluted by adding 14 µL of ultrapure H2O directly to the center of the membrane and centrifuging for 1.5 mins at full speed. Samples were stored at –80°C until initiation of the library preparation procedure.
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| Data processing |
Adapters were trimmed from Fastq files through Flexbar v3.5.
The trimmed Fastq files were processed using paired-end alignment with HISAT2 v2.2.1 and the resulting BAM files were aligned to the mouse reference genome (Mus musculus, GRCm39, Ensemble version 104) for input and IP samples.
Peaks showing significant enrichment in the IP samples vs. corresponding input samples for all submitted replicates were detected using RADAR v0.2.1, with a fragment length of 297 base pairs and bin size of 25 base pairs.
Peak reads coverage for transcripts were visualized with RADAR and the Integrative Genomics Viewer (IGV) browser.
Input mRNA samples were further processed for full decoy-aware transcriptomic analyses using Salmon v1.5.2 on the mouse reference genome (Mus musculus, GRCm39, Ensemble version 104) and processed in R using tximport v1.22.0. Differential gene expression was performed through DESeq2 v1.34.0
Assembly: GRCm39
Supplementary files format and content: CSV format of normalized, filtered, and adjusted reads for IP samples
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| Submission date |
Aug 17, 2022 |
| Last update date |
Aug 24, 2022 |
| Contact name |
Quincy Alexander Hathaway |
| E-mail(s) |
[email protected]
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| Phone |
724-255-4637
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| Organization name |
West Virginia University
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| Street address |
1 Medical Center Drive
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| City |
Morgantown |
| State/province |
WV |
| ZIP/Postal code |
26506 |
| Country |
USA |
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| Platform ID |
GPL30172 |
| Series (2) |
| GSE211479 |
An Adaptive Response through N6-Methyladenosine in Fetal Offspring following Gestational Nano-TiO2 Inhalation Exposure I |
| GSE211481 |
An Adaptive Response through N6-Methyladenosine in Fetal Offspring following Gestational Nano-TiO2 Inhalation Exposure |
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| Relations |
| BioSample |
SAMN30372721 |
| SRA |
SRX17122592 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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