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| Status |
Public on Jul 14, 2016 |
| Title |
Uod-7 (6976) |
| Sample type |
SRA |
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| Source name |
rosette leaves
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| Organism |
Arabidopsis thaliana |
| Characteristics |
cultivar: Uod-7
ecotype id: 6976
developmental stage: before bolting
light condition: long day
tissue: leaf
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| Growth protocol |
Plants were grown in long-day growth chamber at 22 degree celsius.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was extracted from frozen leave tissue from 10 rosettes just before bolting, using the RNeasy Mini Kit according to protocol (QIAGEN).
The mRNA libraries were constructed using the TruSeq Stranded RNA LT Kit (Illumina, San Diego, CA) using 1 - 4 µg total RNA, according to manufacturer's instructions with modifications to confer strand specificity. The mRNA was purified using the RNA Purifications Beads as per Illumina protocol instructions. The mRNA was incubated in the Elute, Prime, Fragment Mix at 94˚C for 8 min. After first strand synthesis (as per Illumina protocol instructions), the product was purified using RNAClean XP beads (Beckman, Brea, CA) as per manufacturer's instructions and eluted in 18 µL nuclease free water. Second strand synthesis was performed by adding the RNAClean XP purified product to 2.5 µL 10x NEB Buffer 2 (New England Biolabs, Ipswich, MA), 2 µL dUTP mix (10mM dATPs, 10mM dGTPs, 10mM dCTPs, and 20mM dUTPs), 0.5 µL RNAse H (2 U/µL), 1 µL DNA Polymerase I (E. coli) (New England Biolabs), and 1 µL DTT (100 mM). The 25 µL mixture was incubated at 16˚C for 2.5 hours. The addition of a 3’ A base and Adapter ligation was performed per Ilumina protocol instructions. The purified ligation products were incubated with 2 µL Uracil DNA Glycosylase (Fermentas) before PCR amplification (also done per Illumina protocol specifications 12 cycles). The reaction products were purified using AMPure XP beads according to manufacturer’s specifications. RNA-seq libraries were sequenced using the Illumina HiSeq 2500 (Illumina) with SE 1X100 Single index read or on HiSeq 4000 with 2X150 PE reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
X6976
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| Data processing |
Base calling was done by RTA version 1.17.20 and 1.18.61. Base call files (bcl) were converted by fastq by CASAVA version 1.8.2.
Sequenced reads were trimmed for adaptor sequence. Read mapping and counting were performed by the rmake pipeline version 1.0c (http://physiology.med.cornell.edu/faculty/mason/lab/r-make/; Sheng Li, Nat Biot 2014).
Reads were mapped to TAIR10 genome assemly and TAIR10 gene models using the STAR aligner version 2.4.2a with parameters "--runThreadN 11 --outFilterMultimapNmax 1 --readFilesCommand zcat --outSAMunmapped Within --outStd SAM". STAR index were generated from TAIR10 genome sequences and TAIR10 gene model GTF file and paramter "--ajdbOverhang 99". Only read1 of the PE reads from HiSeq 4000 were mapped.
Read counts on genes for individual accessions were quantified by the count program in rmake.
To generate the gene by accession expression matrix (supplementary file), raw read counts were first quantile-normalized, then normalized by the RUVg functon in the R package RUVseq version 1.5.0, using a set of 21 reference genes from Wang et al. (Scientific Reports 2014; doi:10.1038/srep06781) with parameter k=4.
Genome_build: TAIR10
Supplementary_files_format_and_content: one tab-delimited text files of raw read counts for each sample
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| Submission date |
Apr 28, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Joseph R Ecker |
| E-mail(s) |
ecker@salk.edu
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| Phone |
8584534100
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| Organization name |
HHMI-Salk-Institute
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| Department |
Genomic Analysis Laboratory
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| Lab |
Ecker lab
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| Street address |
10010 North Torrey Pines Road
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| City |
La Jolla |
| State/province |
CA |
| ZIP/Postal code |
92037 |
| Country |
USA |
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| Platform ID |
GPL17639 |
| Series (1) |
| GSE80744 |
Epigenomic and genome structural diversity in a worldwide collection of Arabidopsis thaliana |
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| Relations |
| BioSample |
SAMN04910464 |
| SRA |
SRX1734582 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2135889_6976.bam.gene.count2.txt.gz |
132.5 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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