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Sample GSM860055 Query DataSets for GSM860055
Status Public on Jan 12, 2012
Title dsrta stat vs WT stat
Sample type RNA
 
Channel 1
Source name sampled in stationary phase
Organism Lactiplantibacillus plantarum
Characteristics strain: dsrta
sample time: stationary phase
Treatment protocol sampled at mid logarythmic and late stationary growth phase
Growth protocol pH controled fermentor culture, 37 degrees, 1.5% glucose
Extracted molecule total RNA
Extraction protocol Quenching Buffer 66.7 mM HEPES (pH 6.5) 60%Methanol Extraction Mixture (Prepared in a screw-cap tube) -500 µl phenol/Chloroform -30 µl10% SDS -30 µl 3 M NaAc (pH 5.2) -500 mg glass-beads (75-150 µm) -400 µl TE buffer 1.For each sample prepare a tube with Extraction Mixture 2.Harvest cells and quench them immediately in Quenching Buffer. Add 4 volumes of Quenching Buffer ( 40°C) to 1 volume of cell culture 3.Mix and store the sample at -40°C 4.Centrifuge cells at -20°C at 9000 rpm for 10 minutes (Centrifuge RC5B) 5.Discard the supernatant; keep the cell pellet cooled and work quickly to prevent condensation of water. Transfer the pellet with a pre-cooled spatula to a screw-crap tube with Extraction Mixture 6.Mix the cell pellet thoroughly with Extraction Mixture by shaking by hand 7.Freeze the tube immediately in liquid nitrogen 8.Break cells in the Savant FastPrep FP120. Three times 40 seconds at speed 4.0 (cool cells between steps if necessary) 9.Centrifuge for 5 minutes at 4°C in an Eppendorf centrifuge at 10.000 rpm 10.Transfer 500 µl of the supernatant to a new tube. Add 400 µl chloroform (chloroform should be cooled at 4°C) 11.Centrifuge for 2 minutes in an Eppendorf centrifuge at full speed (4°C) 12.Continue with the High Pure RNA Isolation Kit from Roche (order # 1 828 665) 13.Incubate for 60 minutes with DnaseI, elute with 50 µl elution buffer 14.Store the RNA in at least 2 aliquots, one of 20 µl (for concentration and quality analysis and labelling) and one back-up of 30 µl at -80°C.
Label Cy5
Label protocol 10 µg of total RNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 3 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy3-label
 
Channel 2
Source name sampled in stationary phase
Organism Lactiplantibacillus plantarum
Characteristics strain: WCFS1
sample time: stationary phase
Treatment protocol sampled at mid logarythmic and late stationary growth phase
Growth protocol pH controled fermentor culture, 37 degrees, 1.5% glucose
Extracted molecule total RNA
Extraction protocol Quenching Buffer 66.7 mM HEPES (pH 6.5) 60%Methanol Extraction Mixture (Prepared in a screw-cap tube) -500 µl phenol/Chloroform -30 µl10% SDS -30 µl 3 M NaAc (pH 5.2) -500 mg glass-beads (75-150 µm) -400 µl TE buffer 1.For each sample prepare a tube with Extraction Mixture 2.Harvest cells and quench them immediately in Quenching Buffer. Add 4 volumes of Quenching Buffer ( 40°C) to 1 volume of cell culture 3.Mix and store the sample at -40°C 4.Centrifuge cells at -20°C at 9000 rpm for 10 minutes (Centrifuge RC5B) 5.Discard the supernatant; keep the cell pellet cooled and work quickly to prevent condensation of water. Transfer the pellet with a pre-cooled spatula to a screw-crap tube with Extraction Mixture 6.Mix the cell pellet thoroughly with Extraction Mixture by shaking by hand 7.Freeze the tube immediately in liquid nitrogen 8.Break cells in the Savant FastPrep FP120. Three times 40 seconds at speed 4.0 (cool cells between steps if necessary) 9.Centrifuge for 5 minutes at 4°C in an Eppendorf centrifuge at 10.000 rpm 10.Transfer 500 µl of the supernatant to a new tube. Add 400 µl chloroform (chloroform should be cooled at 4°C) 11.Centrifuge for 2 minutes in an Eppendorf centrifuge at full speed (4°C) 12.Continue with the High Pure RNA Isolation Kit from Roche (order # 1 828 665) 13.Incubate for 60 minutes with DnaseI, elute with 50 µl elution buffer 14.Store the RNA in at least 2 aliquots, one of 20 µl (for concentration and quality analysis and labelling) and one back-up of 30 µl at -80°C.
Label Cy3
Label protocol 10 µg of total RNA were primed with 2 µl of 100 µM T16N2 DNA primer at 70°C for 10 min, then reversed transcribed at 42°C for 3 h in the presence of 400 U SuperScript II RTase (Invitrogen), and 100 µM each dATP, dTTP, dGTP, with 25 µM dCTP, 25 µM Cy3-label
 
 
Hybridization protocol Agilent hybridization protocol for Two-color microarray-based Gene expression analysis. Version 5.5
Scan protocol Scanned on an ScanArray Express 4000 scanner (Perkin Elmer, Wellesley, MA). Images were quantified using ImaGene Version 7.5 software (BioDiscovery Inc., Marina Del Rey, CA, USA).
Data processing scanned slides were Lowess normalised using Prep (van Hijum, Applied Bioinformatics 2003 2(4):241-244).
 
Submission date Jan 10, 2012
Last update date Jan 12, 2012
Contact name Michiel Wels
E-mail(s) michiel.wels@nizo.com
Organization name NIZO food research
Street address Kernhemseweg 2
City Ede
ZIP/Postal code 6718 ZB
Country Netherlands
 
Platform ID GPL13984
Series (1)
GSE34999 The impact of Lactobacillus plantarum sortase on target-protein sorting, gastrointestinal persistence and host immunomodulation

Data table header descriptions
ID_REF
VALUE Background substracted, Lowess normalised, Cy5/Cy3 ratios (no log transformation), scaled for interslide differences using Postprep

Data table
ID_REF VALUE
1
2 0.168558778
3 1.11030134
4 10.08385626
5 0.766201341
6
7 1.572520101
8
9 0.024386603
10 1.408405061
11 1.438349206
12
13 1.887921571
14
15
16 1.007770661
17
18 1.19716724
19
20 0.857734042

Total number of rows: 9271

Table truncated, full table size 147 Kbytes.




Supplementary file Size Download File type/resource
GSM860055_slide-3_1.txt.gz 240.3 Kb (ftp)(http) TXT
Processed data included within Sample table

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