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| Status |
Public on Jul 30, 2015 |
| Title |
Ler - sample 4 - long fraction |
| Sample type |
SRA |
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| Source name |
Ler_seedling_long fraction
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| Organism |
Arabidopsis thaliana |
| Characteristics |
line background: Ler
genotype/variation: wild type
tissue: seedling
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA were obtained using Phenol:Chloroform extraction. RNA concentrations were measured with the Picodrop and a mix of all RNAs with a concentration of 1 ng/µl was made. 1 µl of this mix was added to each RNA sample before fractionation. The mRNA fractionation was carried out with the Promega PolyATract® System 1000 and the protocol modified as follows:The GTC, DIL, ß-mercaptoethanol (BME), biotinylated oligod(T), 0.5x SSC and H2O were allowed to reach room temperature. Forty-one microliter of BME were added per ml of GTC (GTC/BME) and 20.5 µl BME were added per ml of DIL and preheated to 70 °C. The SSC buffer was diluted to a concentration of 0.085x. In a 2 ml tube, 80 µg of total RNA (in a maximum of 40 µl) were mixed with 400 µl GTC/BME, 15 µl biotinylated oligo d(T) (Promega) and 816 µl DIL/BME and heated to 70 °C for 5 min. Afterwards the samples were spun at 13 000 rpm for 10 minutes at room temperature. In the meantime the paramagnetic beads were washed. Afterwards the beads were resuspended in 600 µl 0.5x SSC. The supernatant of the spun samples was added to the washed beads and the biotinylated oligod(T) was allowed to bind the beads by rotation at room temperature for 15 min. In the following step, the beads were captured and the supernatant transferred to a new tube (unbound fraction). The beads were washed three times with rotation for at least 5 min between each wash step. Afterwards, the beads were resuspended in 400 µl of 0.085 x SSC and rotated for 10 min at room temperature. The beads were capture and the eluate transferred to a new tube (short fraction). This step was repeated once (total of 800 µL). The beads were then washed with 400 µl nuclease free water (rotation for 10 min) twice and the eluates transferred to a new tube (800 µl, long fraction). All collected samples where centrifuged for 10 min at 13000 rpm, 4 °C to remove any transferred beads. Then 0.1 vol of Co-precipitant Pink buffer (BioLine) were added and the samples mixed well. Afterwards 30 µg of Co-precipitant pink (BioLine) were added, mixed well, and 1 vol 100 % ethanol was added. The samples were incubated overnight (15 - 16 hours) at -20 °C. In the next step, the samples were centrifuged at 13000 rpm, 4 °C for 30 min. The supernatant was removed, the pellet washed with 500 µl 80 % ethanol, dried, and dissolved in 15 µl DEPC treated water.
Libraries were prepared using the Illumina TruSeq protocol.
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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 |
Adapter clipping and poly(A)-filtering.
rRNA filtering using RiboPicker (Schmieder et al., 2012).
TopHat2 (Kim et al., 2013) mapping with reference annotation.
Quantifying gene expression using cufflinks (Trapnell et al., 2010).
Genome_build: TAIR10 + spiked-in controls
Supplementary_files_format_and_content: text files containing gene IDs and fpkm values.
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| Submission date |
May 15, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Michael Lenhard |
| E-mail(s) |
michael.lenhard@uni-potsdam.de
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| Phone |
+49-331-9775580
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| Organization name |
Universität Potsdam
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| Department |
Institut für Biochemie und Biologie
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| Street address |
Karl-Liebknecht-Str. 24-25, Haus 26
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| City |
Potsdam |
| ZIP/Postal code |
14476 |
| Country |
Germany |
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| Platform ID |
GPL13222 |
| Series (1) |
| GSE57690 |
Genome-wide analysis of PAPS1-dependent polyadenylation identifies novel roles for functionally specialized poly(A) polymerases in Arabidopsis thaliana |
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| Relations |
| BioSample |
SAMN02778018 |
| SRA |
SRX543026 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1386781_326-Ler-long_R1.fpkm.txt.gz |
185.7 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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