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Sample GSM1876323 Query DataSets for GSM1876323
Status Public on Sep 03, 2018
Title SC-SP_F10DTCtrl_rep1
Sample type RNA
 
Source name SC-SP from the pituitary
Organism Mus musculus
Characteristics gender: Female
genotype: -/iDTR
sorted population: non-sca1high
dt treatment: 10DT
Treatment protocol -/iDTR (ctrl) and GHCre/iDTR female mice were treated with DT for 3 (3DT) or 10 (10DT) days (4 ng / g body weight, twice a day for the indicated number of days).
Extracted molecule total RNA
Extraction protocol Mice were euthanized by exposure to CO2 and the neurointermediate lobe of the pituitary was carefully removed. The AP was isolated and dissociated into single cells using trypsin. Cells were resuspended in pituitary-optimized serum-free defined medium (SFDM; Invitrogen). Dissociated AP cells were incubated with Hoechst33342 (2.5µg/ml; Sigma-Aldrich), stained with phycoerythrin (PE)-conjugated rat anti-mouse stem cell antigen-1 (Sca1; Table 1) (BD Biosciences), incubated with propidium iodide (2µg/ml; Sigma-Aldrich) and analyzed for side population (SP) by FACS (FACSVantage or FACS Aria III; BD Biosciences). To confirm the SP phenotype, verapamil was added (100µM; Sigma-Aldrich) which blocks the SP’s dye efflux. The SP fraction not expressing Sca1 at a high level (non-Sca1high SP) has been identified as the stem cell-clustering population of the pituitary, referred to as the ‘stem cell-SP’ or SC-SP. The SC-SP and the bulk Hoechstmid+high ‘main population’ (MP) were sorted by FACS after 3DT and 10DT treatment into cold lysis solution of the RNeasy Micro kit (Qiagen, Venlo, The Netherlands). Total RNA was extracted immediately after cell collection according to the manufacturer’s protocol, typically starting from 25000-50000 cells per sort. RNA samples were stored at -80°C until further processing for whole-genome microarray
Label Cy3
Label protocol RNA concentration and purity were determined spectrophotometrically using the Nanodrop ND-1000 (Nanodrop Technologies) and RNA integrity was assessed using a Bioanalyser 2100 (Agilent). Per sample, an amount of 1ng of total RNA was amplified and converted to single-stranded antisense cDNA using the NuGEN Ovation Pico WTA System v2.0. All steps were carried out according to the manufacturers protocol (NuGEN).
 
Hybridization protocol 2µg of purified, amplified cDNA were used as input into the Agilent Genomic DNA Enzymatic labeling according to the manufacturers instructions, without the restriction digestion step and then labeled with cyanine 3-dUTP (Cy3). The Cy3-labeled cDNA was quantitated and dye incorporation determined by Nanodrop. 3µg labeled cDNA was hybridized on Mouse Whole Genome v2 Gene Expression 4x44K MicroArray according to the One-Color microarray-Based Gene Expression Analysis User Manual, without fragmentation step.
Scan protocol To assess the raw probe signal intensities, arrays were scanned using the Agilent DNA MicroArray Scanner with surescan High-Resolution Technology and probe signals were quantified using Agilent’s Feature Extraction software (version 10.7.3.1).
Description Gene expression in regenerative female mouse pituitary after 3DT
Data processing Data was base 2 log-transformed and a quantile normalization was applied. Control probes and probes below background were removed.
 
Submission date Sep 14, 2015
Last update date Sep 03, 2018
Contact name Rekin's Janky
E-mail(s) Nucleomics.Bioinformatics@vib.be
Organization name VIB
Department Nucleomics Core
Street address Herestraat 49 Box 816
City Leuven
ZIP/Postal code B-3000
Country Belgium
 
Platform ID GPL11202
Series (1)
GSE72992 Regeneration in the pituitary after cell-ablation injury: time-related aspects and molecular analysis

Data table header descriptions
ID_REF
VALUE Normalized signal intensity averaged by Agilent probe names.

Data table
ID_REF VALUE
A_51_P100034 8.683747234
A_51_P100174 6.509659362
A_51_P100208 6.054771839
A_51_P100289 12.16892822
A_51_P100298 7.162366184
A_51_P100309 1.52718816
A_51_P100327 5.495483886
A_51_P100347 5.366095069
A_51_P100537 2.357209509
A_51_P100573 3.778381793
A_51_P100624 1.531604287
A_51_P100625 1.580932216
A_51_P100768 1.509498103
A_51_P100776 5.296517581
A_51_P100787 7.853245068
A_51_P100828 5.862531051
A_51_P100852 2.335859562
A_51_P100991 7.415905241
A_51_P100997 5.370668169
A_51_P101006 5.876457817

Total number of rows: 38017

Table truncated, full table size 946 Kbytes.




Supplementary file Size Download File type/resource
GSM1876323_14047.txt.gz 8.9 Mb (ftp)(http) TXT
Processed data included within Sample table

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