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Status |
Public on Apr 06, 2021 |
Title |
m6A-RIP-Seq ZCCHC4 KO embryos |
Sample type |
SRA |
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Source name |
C elegans embryos
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Organism |
Caenorhabditis elegans |
Characteristics |
strain: F33A8.4 rip antibody: 250 ug of a-m6A (Cell Signaling Technologies, Cat #56593S) type: RIP
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Extracted molecule |
total RNA |
Extraction protocol |
mRNA was obtained from total RNA (100 ug) by purifying twice with a rRNA depletion kit (RiboMinus Eukaryote Kit v2) following manufacturer’s instructions. m6A RIP-seq experiments were performed as previously described (Sendinc, David-Valle et.al, Molecular Cell, 2019). Briefly: 5 g of mRNA are fragmented at 98ºC for 3 min in 200 ul of fragmenting buffer (10 mM Tris pH 7.4, 10 mM ZnCl2) and quenched with 70mM EDTA. 50 L of Protein G Dynabeads (Invitrogen) are incubated with 250 ug of rabbit monoclonal anti-m6A (Cell Signaling Technologies, Cat #56593S) at 4ºC for 30 mins. The beads/antibody slurry is added to the fragmented mRNA and incubated in rotation for 1 h at 4ºC. Beads were washed twice with 200 L of reaction buffer (150 mM NaCl, 10 mM Tris-HCl, 0.1% Triton X-100, pH 7.5), twice with 200 L of low salt buffer (50 mM NaCl, 10 mM Tris-HCl, 0.1% Triton X-100, pH 7.5) and twice with 200 L of high salt buffer (500 mM NaCl, 10 mM Tris-HCl, 0.1% Triton X-100, pH 7.5). Immunoprecipitated RNA was eluted in TRIzol and purified with the RNA clean and concentrator Kit -5 (Zymo, Cat #R1013). Illumina sequencing libraries are prepared with the NEBNext Ultra II Directional Library Prep Kit for Illumina from NEB (#E7760), following manufacturer's instructions. RNA-Seq libraries are generated from three biological replicates for Input (RNA-Seq) and m6A-immunoprecipitated mRNA. Libraries are sequenced in a NextSeq 550 with an average coverage of 30 million reads per sample.
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Library strategy |
RIP-Seq |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
NextSeq 550 |
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Description |
ce11-m6A-ZCCHC4_KO-A-lfc.bw ce11-m6A-ZCCHC4_KO-B-lfc.bw ce11-m6A-ZCCHC4_KO-C-lfc.bw
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Data processing |
Reads were aligned with hisat2 (v2.0.4, parameters: --no-unal --rna-strandness R) using the gtf annotation WBcel235 downloaded from Ensembl genes v96 as reference. Sam files were converted to bam files using samtools (v 1.9, parameters: -bSq 10). Bigwig files were generated using the bamCoverage program from the deepTools package (v3.3.0, parameters: -bs 20 --normalizeUsing BPM --skipNAs --ignoreDuplicates). Gene counts for the Input files (RNAseq experiments) were obtained with the featureCounts program fron the Rsubread package (v 1.34.1, parameters minMQS=10, allowMultiOverlap=F, largestOverlap=T, strandSpecific=2) using the WBcel235 annotation from Ensembl genes v96. Normalization and dispersion factors for RNAseq analysis were calculated using the functions calcNormFactors() and estimateDisp() from the edgeR package (v 3.26.1). DEG were calculated using the glmFit() and glmLRT() functions from edgeR. Genes with a |logFC| > 1 and FDR < 1e-5 were considered differentially expressed. m6A peaks were calculated with the R package exomePeaks (v 2.17) using a FDR cutoff of 1e-10 and a fold enrichment of 10. Only consistent peaks (identified in all replicates) were included. Peaks within genes with low expression level (cpm < 1) were ignored. Genome_build: WBcel235 Supplementary_files_format_and_content: Log-fold change m6A/Input bigwig files were generated using bamCoverage from the deepTools package (v3.3.0, parameters: -bs 20 --normalizeUsing BPM --skipNAs --ignoreDuplicates) Supplementary_files_format_and_content: log2 TPM for RNAseq is generated from normalized counts estimated by edgeR with the rpkm() function Supplementary_files_format_and_content: BED file containing the estimated m6A peaks for N2 embryos is generated from the output of exomePeaks, aplying the cut-offs values as described
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Submission date |
Apr 16, 2020 |
Last update date |
Apr 06, 2021 |
Contact name |
David Valle-Garcia |
Organization name |
National Autonomous University of Mexico
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Department |
Institute of Biotechnology
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Street address |
Av Universidad 2001 Col Chamilpa
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City |
Cuernavaca |
State/province |
Morelos |
ZIP/Postal code |
62210 |
Country |
Mexico |
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Platform ID |
GPL27998 |
Series (1) |
GSE148786 |
RNA m6A methylome of Caenorhabditis elegans |
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Relations |
BioSample |
SAMN14605533 |
SRA |
SRX8123983 |
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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