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Sample GSM2264187 Query DataSets for GSM2264187
Status Public on Aug 08, 2019
Title luh4_4h
Sample type SRA
 
Source name roots_with Al
Organism Arabidopsis thaliana
Characteristics tissue: root
old of seedlings: six-days
time point: 4h
genotype/variation: luh-4 mutant
treament: with Al
ecotype: Columbia
Treatment protocol Approximately 500 seedlings (6-d-old) of both the wild-type Col and luh-4 mutant line were exposed to a nutrient solution containing 0 or 25 μM AlCl3 (pH4.2). After 4 h, the roots were sampled for RNA isolation.
Growth protocol The seedlings were pre-grown in nutrient solution for 6 d at pH5.5 in a growth chamber with a 16/8 h light/dark cycle at 22 oC.
Extracted molecule total RNA
Extraction protocol Total RNA was isolated using the RNeasy® Plant Mini Kit (QIAGEN GmbH, Qiagen, GmbH, Hilden, Germany) following the manufacturer’s protocol. RNA libraries were prepared for sequencing using standard Illumina protocols
The total RNA samples are first treated with DNase I to degrade any possible DNA contamination. Then the mRNA is enriched by using the oligo(dT) magnetic beads (for eukaryotes). Mixed with the fragmentation buffer, the mRNA is fragmented into short fragments (about 200 bp). Then the first strand of cDNA is synthesized by using random hexamer-primer. Buffer, dNTPs, RNase H and DNA polymerase I are added to synthesize the second strand. The double strand cDNA is purified with magnetic beads. End reparation and 3'-end single nucleotide A (adenine) addition is then performed. Finally, sequencing adaptors are ligated to the fragments. The fragments are enriched by PCR amplification. During the QC step, Agilent 2100 Bioanaylzer and ABI StepOnePlus Real-Time PCR System are used to qualify and quantify of the sample library. The library products are ready for sequencing via Illumina HiSeqTM 2000 or other sequencer when necessary.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
 
Data processing By base calling, the original image data produced by the sequencer is transferred into sequences, which are defined as "raw reads"(or "raw data") and saved as ".fastq" files. As the raw reads may contain low quality reads and or adaptor sequences, preprocessing is necessary before starting further analysis. Normally, data cleaning (or data filtering) is performed to obtain "clean reads" (or "clean data") for further analysis.
As there are some adaptor sequences and/or low quality reads present in the raw reads, data filtering is carried out to obtain high quality reads as the clean reads (clean data). The procedure include following steps: 1. Remove reads with adaptor sequences. 2. Remove reads in which the percentage of unknown bases (N) is greater than 10%. 3. Remove low quality reads. If the percentage of the low quality base (base with quality value ≤ 5) is greater than 50% in a read, we define this read as low quality
The gene expression level is calculated by using RPKM[Mortazavi et al. (2008). Nature Methods. 5(7): 621-8] method (Reads Per kb per Million reads),
Clean reads were mapped to reference sequences and/or reference gene set using SOAPaligner/SOAP2[Li R et al. (2009) Bioinformatics, 25 (15). 1966-1967.]. No more than 2 mismatches were allowed in the alignment
Genome_build: TAIR10
Supplementary_files_format_and_content: Excel files include RPKM values for each Sample
 
Submission date Aug 08, 2016
Last update date Aug 08, 2019
Contact name Zhong-Bao Yang
E-mail(s) zbyang@sdu.edu.cn
Organization name Shandong University (Qingdao)
Department School of Life Science
Lab he Key Laboratory of Plant Development and Environmental Adaptation Biology, Ministry of Education
Street address 72# Binhai Road
City Qingdao
State/province Shandong
ZIP/Postal code 266237
Country China
 
Platform ID GPL13222
Series (1)
GSE85292 The luh-4 mutant has a differential transcriptional program in response to Al stress
Relations
BioSample SAMN05521267
SRA SRX2007316

Supplementary file Size Download File type/resource
GSM2264187_luh4_4h.Gene.rpkm.txt.gz 1.9 Mb (ftp)(http) TXT
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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