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Sample GSM4448882 Query DataSets for GSM4448882
Status Public on Mar 31, 2021
Title Holstein Friesian R microplus larval feeding replicate 1
Sample type RNA
 
Channel 1
Source name Holstein Friesian superficial cervical lymph node, R microplus larval feeding
Organism Bos taurus
Characteristics tissue: Lymph node
breed: Holstein
sample type: Rhipicephalus microplus larvae tick attachment
Treatment protocol Lymph node biopsies were collected, kept on ice and transferred to an RNA stabilizing solution (0.5 M EDTA, 1 M ammonium citrate, 5.3 M ammonium sulphate, adjusted to pH 5.2 with H2SO4).
Extracted molecule total RNA
Extraction protocol Total RNA was isolated using TRI-reagent fractionation (Sigma-Aldrich) and the RNeasy mini kit (QIAGEN, Germany) following the manufacturer’s instructions. Contaminating genomic DNA was removed with a DNase I treatment (QIAGEN, Germany).
Label Cy5
Label protocol First-strand cDNA synthesis was performed by incubating 4 µg RNA (from each biological replicate and the reference pool) with 250 pmol oligo(dT25) and 775 pmol random primer 9 for 10 minutes at 70˚C, followed by cooling on ice for 10 minutes. Reverse transcription and aminoallyl-dUTP (5-(3-aminoallyl)-2’deoxyuridine-5’ triphosphate) incorporation were performed simultaneously using 340 units SuperScript® III reverse transcriptase (Invitrogen™ life technologies, USA).
cDNA samples were coupled to 75 picomol Cy3 (reference pool) and Cy5 (samples) fluorescent dyes (GE Healthcare Life Sciences)at pH 9 dissolved in DMSO. Unincorporated dye was then removed.
 
Channel 2
Source name Pooled cattle superficial cervical lymph nodes
Organism Bos taurus
Characteristics tissue: Lymph node
Treatment protocol Lymph node biopsies were collected, kept on ice and transferred to an RNA stabilizing solution (0.5 M EDTA, 1 M ammonium citrate, 5.3 M ammonium sulphate, adjusted to pH 5.2 with H2SO4).
Extracted molecule total RNA
Extraction protocol Total RNA was isolated using TRI-reagent fractionation (Sigma-Aldrich) and the RNeasy mini kit (QIAGEN, Germany) following the manufacturer’s instructions. Contaminating genomic DNA was removed with a DNase I treatment (QIAGEN, Germany).
Label Cy3
Label protocol First-strand cDNA synthesis was performed by incubating 4 µg RNA (from each biological replicate and the reference pool) with 250 pmol oligo(dT25) and 775 pmol random primer 9 for 10 minutes at 70˚C, followed by cooling on ice for 10 minutes. Reverse transcription and aminoallyl-dUTP (5-(3-aminoallyl)-2’deoxyuridine-5’ triphosphate) incorporation were performed simultaneously using 340 units SuperScript® III reverse transcriptase (Invitrogen™ life technologies, USA).
cDNA samples were coupled to 75 picomol Cy3 (reference pool) and Cy5 (samples) fluorescent dyes (GE Healthcare Life Sciences)at pH 9 dissolved in DMSO. Unincorporated dye was then removed.
 
 
Hybridization protocol Equivalent picomoles (20 pmol) of Cy3-labelled cDNA from the common reference pool were hybridised with Cy5-labelled individual test cDNA. Overnight hybridisation at 65°C (rotation speed of 10), washing and post-processing were performed at the ACGT Microarray Facility (University of Pretoria, South Africa). Prior to slide scanning with the Axon GenePix 4000B scanner (Molecular Devices), slides were washed and dried by centrifugation
Scan protocol Scanned on an Axon GenePix 4000B scanner (Molecular Devices)
Images were measured, recorded, analysed and spots were inspected using Axon GenePix Pro 6.0 software (Molecular Devices)
Description Biological replicate 1 of 3. Holstein Friesian lymph node tissue. Rhipicephalus microplus larvae tick attachment.
Data processing Axon GenePix Pro 6.0 software (Molecular Devices) and the linear model for microarray data analysis (LIMMA) within the R statistical environment (http://cran.r-project.org/) was employed. Adaptive background correction (offset = 50) was followed by within-array normalisation (Robust Spline) and between-array normalisation (G quantile). Fold change was determined between all transcripts within a cattle breed collected at different time-points using the empirical Bayesian statistics, which were subsequently expressed as P- values (corrected for false discovery rate).
 
Submission date Apr 01, 2020
Last update date Mar 31, 2021
Contact name Dave Kenneth Berger
E-mail(s) dave.berger@up.ac.za
Phone +27124204634
Organization name University of Pretoria
Department Plant and Soil Science
Lab Molecular and Plant Pathogen Interactions
Street address Lynnwood Road
City Pretoria
State/province Gauteng
ZIP/Postal code 0002
Country South Africa
 
Platform ID GPL11648
Series (1)
GSE147918 Cattle lymph nodes: Control vs. Larva tick-infested; Control vs. Adult tick-infested; Larva tick-infested vs. Adult tick-infested

Data table header descriptions
ID_REF
VALUE Robust Spline-normalized, Gquantile-normalized log2 (Cy5/Cy3) ratios representing test/reference

Data table
ID_REF VALUE
1 -0.101465233
2 0.058262004
3 0.091110147
4 -0.057525572
5 -0.067997325
6 0.028119228
7 0.022100921
8 0.081739685
9 0.046265507
10 0.376113681
11 0.093308925
12 -0.176389555
13 0.198301066
14 0.040346845
15 -0.092384826
16 0.061289251
17 0.028251471
18 -0.346853727
19 0.110438302
20 -0.54815453

Total number of rows: 45220

Table truncated, full table size 801 Kbytes.




Supplementary file Size Download File type/resource
GSM4448882_HFries_T1_rep1.gpr.gz 3.9 Mb (ftp)(http) GPR
Processed data included within Sample table

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