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Sample GSM4478911

Query DataSets for GSM4478911

Status

Public on Apr 06, 2021

Title

RNAseq ZCCHC4 KO embryos

Sample type

SRA

Source name

C elegans embryos

Organism

Caenorhabditis elegans

Characteristics

strain: F33A8.4
type: Input

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.

Library strategy

RIP-Seq

Library source

transcriptomic

Library selection

other

Instrument model

NextSeq 550

Description

ce11-embryos-RNA_seq-TPM_log2.txt

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

Submission date

Apr 16, 2020

Last update date

Apr 06, 2021

Contact name

David Valle-Garcia

Organization name

National Autonomous University of Mexico

Department

Institute of Biotechnology

Street address

Av Universidad 2001 Col Chamilpa