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Sample GSM5049385

Query DataSets for GSM5049385

Status

Public on Aug 11, 2021

Title

RNA-seq Nipp root rep1

Sample type

SRA

Source name

WT rice root

Organism

Oryza sativa

Characteristics

ecotype: Nipponbare (Nipp)
genotype: wild type
ip antibody: none
tissue: root

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

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

non-ribosomal RNA
root_gene_featureCounts.txt
root_repeat_featureCounts.txt

Data processing

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