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| Status |
Public on Jun 20, 2014 |
| Title |
No-0_24hr |
| Sample type |
SRA |
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| Source name |
Leaf, wild type, 24hr
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| Organism |
Arabidopsis thaliana |
| Characteristics |
ecotype: Nossen (No-0)
genotype/variation: wild type
tissue: leaf
age: 4 weeks
treatment: 21 °C
timepoint: 24 h at 21°C
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| Treatment protocol |
Arabidopsis No-0 and slh1 plants were grown for 4 weeks at 28°C after germination on MS plate. Plants were transferred to 21°C growth chamber at the beginning of the light cycle and samples were harvested at 0h, 6 h, 9 h and 24 h after transfer for total RNA extraction.
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| Growth protocol |
Arabidopsis thaliana (No-0 and slh1) plants were grown in short day conditions (10 h light / 14 h dark) for 4 weeks at 28°C after germination on MS plate.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted using the TRI reagent (Sigma) and 1-Bromo-3-chloropropane (Sigma), as per manufacturer's guidelines. RNA was precipitated with half volume of isopropanol and half volume of high salt precipitation buffer (0.8 M sodium citrate and 1.2 M sodium chloride). RNA samples were treated with DNaseI (Roche) according to the manufacturer's recommendation and phenol/chloroform extracted and ethanol precipitated.
Typically, 5 µg of total RNA was used to generate first strand cDNA using a oligo(dT) primer comprising P7 sequence of Ilumina flowcell. Double strand cDNA is synthesized as described previously [Okayama & Berg, Molecular and cellular biology 1982, 2:161-170]. Purified cDNA is subjected to Covaris shearing to a target size of 200bp (parameters: Intensity - 5, Duty cycle - 20%, Cycles/Burst - 200, Duration - 90seconds). End repairing and A-tailing of sheared cDNA is carried out as described by Illumina. Y-shaped adapters are ligated to A-tailed DNA and subjected to size selection on 1X TAE agarose gel. The gel-extracted library is PCR enriched and quantified using qPCR with previously sequenced similar size range Illumina library.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina MiSeq |
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| Data processing |
Library strategy: Tag-Seq
Illumina Casava1.7 software used for basecalling.
Illumina sequence library is quality filtered using FASTX Toolkit 0.0.13 with parameters -q20 and -p50 and reads containing "N" are discarded.
EXPRSS libraries are separated using perfect match to the barcode (at 5' end). Each sub-library is quality filtered (-q20 and -p50) and artifact filtered using FASTX-toolkit.
Quality filtered library was aligned to the Arabidopsis thaliana Col-0 genome sequences (TAIR10) using Bowtie version 0.12.8 (-a -m 10 --best --strata) and selected reads with up to 10 reportable alignments. Unaligned reads from previous step are used to align to transcript sequences of Arabidopsis using Novoalign V2.08.03 (-s 1 -r A 100), since reads are from No-0 background. Tag to gene association is carried out using following considerations. Reads aligning in sense orientation with in each gene limits are assigned to that gene. A read aligning to all splice variant of a gene is counted once, and the reads uniquely aligning to various splice variants of a gene are pooled. Reads aligning to genes with overlapping gene limits are split equally between them. Reads aligning to more than 10 genes are discarded. Reads aligning to up to 10 genes are split equally between them. Reads aligning on the anti-sense strand of any gene are tested for if the read falls within 500bp from 3' end of another gene in sense direction. Otherwise reads are assigned as antisense tags for the specific gene. The tag assignment process was implemented in perl and scripts are available on request. Differential expression analysis was performed using the R statistical language version 2.15.3 with the Bioconductor package baySeq version 1.12.0 using 10000 iterations to estimate empirical distribution on the parameters of the Negative Binomial distribution.
Genome_build: TAIR10
Supplementary_files_format_and_content: Tab-delimited text files containing each genes tag counts for individual samples.
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| Submission date |
Nov 20, 2013 |
| Last update date |
May 15, 2019 |
| Contact name |
Ghanasyam Rallapalli |
| E-mail(s) |
g.rallapalli@uea.ac.uk
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| Organization name |
The Sainsbury Laboratory
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| Street address |
Norwich Research Park
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| City |
Norwich |
| ZIP/Postal code |
NR4 7UH |
| Country |
United Kingdom |
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| Platform ID |
GPL17970 |
| Series (2) |
| GSE51721 |
EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics |
| GSE52596 |
EXPRSS: an Illumina based high-throughput expression-profiling method to reveal transcriptional dynamics - III |
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| Relations |
| BioSample |
SAMN02415929 |
| SRA |
SRX380954 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1272333_Read1_No-0_24hr_36bp.txt.gz |
96.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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