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Sample GSM5059748 Query DataSets for GSM5059748
Status Public on Oct 05, 2021
Title Bodenlos_mutant_rep3
Sample type RNA
 
Source name Bodenlos_mutant
Organism Arabidopsis thaliana
Characteristics ecotype background: Columbia (Col-0)
genotype/variation: Bodenlos (IAA12) mutant
tissue: 8-cell embryos
Treatment protocol Ovules were collected from ~60 siliques using vacuum extraction. Siliques were stuck to double-sided tape and sliced open using a needle. Open siliques were submerged in 1x PhosphateBuffered Saline (PBS) buffer and ovules were collected using a vacuum pump through 50 µm filters. Collected ovules were then transferred to Isolation buffer (1x First Strand Buffer (FSB; Invitrogen), 1mM Dithiotreitol (DTT), 4% RNAseLater, MQ), and volume was reduced to ~20 µL. Embryo isolation was performed according to (Raissig, 2013) with the following adaptations. A Zeiss Confocor 1 inverted microscope (Carl Zeiss Microscopy GmbH, Jena, Germany) together with an Eppendorf Transferman 4r micromanipulator (Eppendorf AG) and VacuTip II microcapillaries (Eppendorf) were used to isolate about 40-50 washed embryos in 50 μl isolation buffer.
Growth protocol All seeds were sterilized in 25% bleach/75% ethanol solution for 10 minutes and were afterwards washed twice with 70% ethanol and once with 100% ethanol. Dried seed were plated on half-strength Murashige and Skoog (MS) medium and the appropriate antibiotic (in concentration: 50 mg/l kanamycin or 15 mg/l phosphinothricin) for selection of transgenic seeds. After 24hours incubation at 4°C, the plants were grown under long-day conditions at 22°C in a growth room.
Extracted molecule total RNA
Extraction protocol Total RNA was isolated using Trizol-Chloroform extraction followed by clean-up using the RNeasy Plant Mini kit from Qiagen. RNA integrity was checked on chip analysis (Agilent 2100 Bioanalyzer, Agilent Technologies, Amsterdam, The Netherlands) according to the manufacturer's instructions. RNA was judged as suitable for array hybridization only if samples exhibited intact bands corresponding to the 18S and 28S ribosomal RNA subunits, and displayed no chromosomal peaks or RNA degradation products (RNA Integrity Number > 8.0).
Label biotin
Label protocol One hundred nanogram of RNA was used for whole transcript cDNA synthesis with the Ambion WT expression kit [catalog number 4411974] (Applied Biosystems/Life Technologies, Nieuwekerk a/d IJssel, The Netherlands).
 
Hybridization protocol Hybridization of 5.5μg labelled cDNA was done overnight for 17 hours, at 60 rpm, at 45ºC in a Hybridization Oven 640 (Affymetrix). The protocol was conducted as described in the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay Manual, chapter 5 (P/N 701880, revision 5). Washing and staining of the arrays were done on an Affymetrix 450 Fluidics Station using the protocolFS450_0002, as described in the Affymetrix Whole Transcript (WT) Sense Target Labeling Assay Manual, chapter 5 (P/N 701880, revision 5).
Scan protocol Arrays were scanned on an Affymetrix 3000 7G scanner, as described in the GeneChip Expression Analysis Technical Manual, section 2 (Eukaryotic Sample and Array Processing), chapter 2 (Eukaryotic Arrays: Washing, Staining and Scanning (P/N 701028, revision 5).
Data processing Expression estimates were calculated applying the RMA algorithm in the Bioconductor library 'affy' (v1.44).
 
Submission date Feb 02, 2021
Last update date Oct 05, 2021
Contact name Dolf Weijers
E-mail(s) dolf.weijers@wur.nl
Organization name Wageningen University
Department Biochemistry
Street address Stippeneng 4
City Wageningen
ZIP/Postal code 6708WE
Country Netherlands
 
Platform ID GPL22599
Series (1)
GSE165986 A molecular framework for control of oriented cell division in the Arabidopsis embryo

Data table header descriptions
ID_REF
VALUE RMA values

Data table
ID_REF VALUE
AT1G01010_at 3.62044516060323
AT1G01020_at 82.1710679535657
AT1G01030_at 3.39941688893851
AT1G01040_at 17.2421773714561
AT1G01046_at 4.16390504132009
AT1G01050_at 405.71816265624
AT1G01060_at 7.00263781385311
AT1G01070_at 4.1153045259844
AT1G01073_at 2.36870830795254
AT1G01080_at 83.6665500234896
AT1G01090_at 1419.66853407183
AT1G01100_at 271.177249090246
AT1G01110_at 9.84214259037342
AT1G01115_at 3.81329698670468
AT1G01120_at 7.07013668312367
AT1G01130_at 5.16075859875887
AT1G01140_at 11.6154428353333
AT1G01150_at 3.88158517605463
AT1G01160_at 547.63907391511
AT1G01170_at 97.2859074179985

Total number of rows: 27826

Table truncated, full table size 812 Kbytes.




Supplementary file Size Download File type/resource
GSM5059748_G279_G05_07_RB4.CEL.gz 4.7 Mb (ftp)(http) CEL
Processed data included within Sample table

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