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| Status |
Public on May 15, 2018 |
| Title |
hspm1mace |
| Sample type |
SRA |
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| Source name |
post-meiotic heat stress
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| Organism |
Solanum lycopersicum |
| Characteristics |
tissue: pollen developmental stage
stress: heat stress
cultivar: Red Setter
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| Extracted molecule |
polyA RNA |
| Extraction protocol |
Pollen was isolated from anthers by squeezing the stamens with a pipette tip 200 μl in the germination solution, followed by vortexing for 15 seconds. Pollen was passed through gauze cloth, washed twice with germination solution, and centrifuged at 4°C for 2 min at 100x g, followed by short spin to sediment the isolated pollen grains. The pellet was resuspended in 200 μl of germination solution, followed by centrifugation at 100 g for 2 minutes at 4°C, and followed by short spin. The supernatant was discarded and samples preserved in the liquid nitrogen. RNA was isolated from two biological replicates per developmental stage and condition using the Macherey-Nagel NucleoSpin miRNA isolation kit according to manufacturer’s protocol. From the two resulting fractions the one comprising the large RNA (> 200 nt) was used for the next steps.
The MACE was carried out according to the protocol described by Zawada et al. 2014 (PMID: 24184689): MACE libraries were prepared using the proprietary TrueQuant technology for elimination of PCR bias (Zawada et al. 2014). Poly-adenylated RNA was extracted with Dynabeads mRNA Purification kit (Life Technologies) from large RNA (>200 nt) and reverse transcribed with SuperScript Double-Stranded cDNA Synthesis Kit (Life Technologies) using biotinylated poly(dT) primers. Fragmentation of cDNA was conducted with Bioruptor (Diagenode) which yielded an average size of 250 bp. Biotinylated cDNA ends were captured by Dynabeads M-270 Streptavidin Beads (Life Technologies) and ligated with T4 DNA Ligase 1 (NEB) to modified adapters (TrueQuant, GenXPro). PCR amplification was performed with KAPA HiFi Hot-Start Polymerase (KAPA Biosystems), purified by Agencourt AMPure XP beads (Beckman Coulter) and sequenced with HiSeq2000 (Illumina)
MACE (massive analysis of cDNA ends)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2000 |
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| Data processing |
FastQC preprocessing
reads were aligned by NextGenMap (version 0.4.12, Sedlazeck et al. 2013) using the following alterations of default parameters: --kmer-skip 1, --silent-clip and --no-unal
Read counts were determined for each annotated gene using HTSeq
TPM (Transcripts Per Million) values were calculated for each gene by dividing the read count of a gene by the total number of aligned reads within the library and multiplying it with one million. Next, all TPM values below a value of 5 were set to zero. The cut-off of 5 was determined by analyzing the expression of genes which are not expected to be detected in pollen.
Genome_build: Solanum lycopersicum cv. Heinz version SL2.50
Supplementary_files_format_and_content: 12 csvfiles (.csv) representing the read counts of all genes in the samples. 1 csvfile containing the TPM values for all genes in the 12 samples (cut-off of 5 already applied).
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| Submission date |
Jan 25, 2018 |
| Last update date |
Jul 18, 2018 |
| Contact name |
Stefan Simm |
| Organization name |
University Medicine Greifswald
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| Department |
Bioinformatics
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| Lab |
Applied Bioinformatics
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| Street address |
Walter-Rathenau-Straße 48
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| City |
Greifswald |
| ZIP/Postal code |
17489 |
| Country |
Germany |
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| Platform ID |
GPL16345 |
| Series (1) |
| GSE109672 |
The coupling of transcriptome and proteome adaptation during development and heat stress response of tomato pollen |
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| Relations |
| BioSample |
SAMN08397382 |
| SRA |
SRX3600899 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2948299_hspm1mace_read_counts.csv.gz |
159.0 Kb |
(ftp)(http) |
CSV |
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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