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Sample GSM5049416

Query DataSets for GSM5049416

Status

Public on Aug 11, 2021

Title

m6A-seq FTO mutant shoot IP rep2

Sample type

SRA

Source name

FTO transgenic mutant rice shoot

Organism

Oryza sativa

Characteristics

ecotype: Nipponbare (Nipp)
genotype: FTO homogeneous transgenic mutant
ip antibody: anti-m6A (NEB, monoclonal, rabbit,Cat. No. E1615S)
tissue: shoot

Growth protocol

Nipp and FTO rice were grown in Kimura B solutionKimura B solution a 12-h light (26 °C)/12-h dark (22 °C) photoperiod, 50–60% relative humidity and 700 μmol m-2 s-1 light. 15 days old plants were harvested for experiment

Extracted molecule

polyA RNA

Extraction protocol

For RNA-seq, 15-day-old seedlings of the Nipp and FTO plant were used for sequencing. Before purified non-ribosomal RNA with Ribo-Zero rRNA Removal Kit (Illumina), ERCC RNA spike-in control (Ambion) was added to each sample (0.1 μl per 5 ug total RNA). Library preparation was performed by using the NEBNext® Ultra™ I Directional RNA Library Prep Kit for Illumina® (NEB #E7420S) according to the manufacturer’s protocols. Sequencing was performed on an Illumina HiSeq machine in pair-end mode with 150 bp per read (Genewiz company, China). For m6A-seq, 1 g of 15-day-old seedlings of the Nipp, FTOmut and FTO plants from hydroponic experiments were used to isolate poly(A) RNA for m6A sequencing. 5 pg spike-ins for m6A seq mixture containing 3 diverse 20% m6A modified and 3 diverse non-m6A modified poly(A) spike-ins (see Supplementary Table 6) which synthesized in vitro used the MEGAscript ® Kit (AM1333, Thermo Fisher Scientific, USA) was added to the poly(A) RNA, followed by fragmented into ~100 nt using RNA Fragmentation Reagents (Ambion, USA) according to the manufacturer’s protocol. m6A-IP was performed using EpiMark N6-Methyladenosine Enrichment Kit following the manufacturer’s protocol. Library preparation was performed by using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB, USA) according to the manufacturer’s protocol. Sequencing was performed on an Illumina HiSeq 4000 machine in pair-end mode with 150 bp per read (Genewiz, China).
For RNA-seq, library preparation was performed by using the NEBNext® Ultra™ I Directional RNA Library Prep Kit for Illumina® (NEB #E7420S) according to the manufacturer’s protocols. Sequencing was performed on an Illumina HiSeq machine in pair-end mode with 150 bp per read (Genewiz company, China). For m6A-seq, library preparation was performed by using NEBNext® Ultra™ II Directional RNA Library Prep Kit for Illumina® (NEB #E7760S) according to the manufacturer’s protocols. Sequencing was performed on an Illumina HiSeq machine in pair-end mode with 150 bp per read (Genewiz company, China).

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 4000

Description

FTOmut_shoot_rep2_gene_MeTPeak.txt
FTOmut_shoot_rep2_repeat_MeTPeak.txt

Data processing

Library strategy: m6A-seq
Adapters of raw reads were clipped by Cutadapt v1.15. Reads longer than 15 nt after trimming were mapped to the rice genome (MSU 7.0) using HISAT2 v2.1.0 with default parameters. mRNA and repeat annotation were downloaded from the Rice Genome Annotation Project and the Rice Annotation Project (RAP-DB), respectively.
For RNA-seq, reads on genes were counted by featureCounts (-t exon -g gene_id -C -M -O --fraction -T 24 -s 2 -p) from from Subread v1.6.4.
For m6A-seq, mapped reads were separated by strands and then MeTPeak was used to detect m6A peaks respectively with the mRNA and repeat anotation (FRAGMENT_LENGTH=200, READ_LENGTH=150, PEAK_CUTOFF_FDR=0.05, WINDOW_WIDTH=50, SLIDING_STEP=10, FOLD_ENRICHMENT=2).
Genome_build: MSU 7.0
Supplementary_files_format_and_content: Raw counts and peaks of mRNAs and repeats were present in tab-delimited plain text files

Submission date

Jan 28, 2021

Last update date

Aug 11, 2021

Contact name

Shun Liu

Organization name

University of Chicago

Department

Chemistry

Lab

Chuan He Lab