|
| Status |
Public on Aug 15, 2019 |
| Title |
granulosa cells_sownr2673_follicle3_largefollicle_LowVAR_LowCOChealth |
| Sample type |
RNA |
| |
|
| Channel 1 |
| Source name |
granulosa cells
|
| Organism |
Sus scrofa |
| Characteristics |
origin: TopigsNorsvin N-line, multiparous sows
follicle size: large
var: low
coc health: low
|
| Treatment protocol |
A total of 29 multiparous Dutch Landrace sows (parity 3 to 5; Topigs Norsvin, Vught, the Netherlands) were used. The sows were fed a standard lactation diet (ca. 12.5MJ NE/kg, 154 g/kg CP, 9.3 g/kg lysine; Lacto Excellent, Agrifirm, Apeldoorn, The Netherlands). Within 24 hours after parturition, piglets were cross-fostered to ensure 13 suckling piglets per sow. The sows had a lactation period of 26.1±0.2 (25 to 27) days and weaned 13.0±0.9 piglets. The sows were slaughtered at the slaughterhouse by stunning and exsanguination within 2 hours after weaning.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from granulosa cells isolated using laser capture micro-dissection (Arcturus) and the Picopure RNA Isolation kit (Arcturus), quantified using Nanodrop (IsoGen Life Science) and qualified using TapeStation (Agilent).
|
| Label |
Cy5
|
| Label protocol |
Approximately 40 ng of purified individual total RNA was used for cDNA synthesis, splitted in two equal fractions and used for subsequent cRNA labeling and synthesis using Cy5 and Cy3 dyes and the Agilent low RNA input fluorescent lineair amplification protocol, as described previously by van Schothorst et al. (Anal Biochem 2007;363:315-7). 32 Cy3-labeled samples with the highest cRNA yield were used for the reference pool.
|
| |
|
| Channel 2 |
| Source name |
granulosa cells
|
| Organism |
Sus scrofa |
| Characteristics |
origin: TopigsNorsvin N-line, multiparous sows
sample type: reference
|
| Treatment protocol |
A total of 29 multiparous Dutch Landrace sows (parity 3 to 5; Topigs Norsvin, Vught, the Netherlands) were used. The sows were fed a standard lactation diet (ca. 12.5MJ NE/kg, 154 g/kg CP, 9.3 g/kg lysine; Lacto Excellent, Agrifirm, Apeldoorn, The Netherlands). Within 24 hours after parturition, piglets were cross-fostered to ensure 13 suckling piglets per sow. The sows had a lactation period of 26.1±0.2 (25 to 27) days and weaned 13.0±0.9 piglets. The sows were slaughtered at the slaughterhouse by stunning and exsanguination within 2 hours after weaning.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated from granulosa cells isolated using laser capture micro-dissection (Arcturus) and the Picopure RNA Isolation kit (Arcturus), quantified using Nanodrop (IsoGen Life Science) and qualified using TapeStation (Agilent).
|
| Label |
Cy3
|
| Label protocol |
Approximately 40 ng of purified individual total RNA was used for cDNA synthesis, splitted in two equal fractions and used for subsequent cRNA labeling and synthesis using Cy5 and Cy3 dyes and the Agilent low RNA input fluorescent lineair amplification protocol, as described previously by van Schothorst et al. (Anal Biochem 2007;363:315-7). 32 Cy3-labeled samples with the highest cRNA yield were used for the reference pool.
|
| |
|
| |
| Hybridization protocol |
Individual Cy5-labelled samples were hybridized against the Cy3-labelled reference pool according to the manufacturers' procedure using Agilent Gene Expression Hybridization Kit. Samples were applied to 8*60K microarrays enclosed in Agilent SureHyb hybridization chambers and hybridized at 65C for 17 hours at 10rpm rotation. After hybridization, slides were washed sequential following Agilents' recommendations and finally covered with an ozone-barrier slide.
|
| Scan protocol |
Scanned with an Agilent Technologies Scanner G2505B with 10 and 100% laser-power intensities.
|
| Description |
Cy3 samples were pooled on a equimolar basis and served as reference pool, and individual Cy5-labelled samples were hybridized against the reference pool.
|
| Data processing |
Images were quantified using Agilent Feature Extraction Software (10.7.3.1) to obtain raw median signal and background values for both Cy5 and Cy3. QC checks were done on raw data and all arrays passed. Spots with a mean signal higher than twice the background value over all arrays and both channels were considered to be expressed. Data was normalized according to Pellis et al. (Physiol Genomics 2003; 16:99-106) based on the Cy3-reference pool, and log2 transformation.
|
| |
|
| Submission date |
Jan 16, 2019 |
| Last update date |
Aug 15, 2019 |
| Contact name |
Evert M. van Schothorst |
| E-mail(s) |
evert.vanschothorst@wur.nl
|
| Organization name |
Wageningen University
|
| Lab |
Human and Animal Physiology
|
| Street address |
De Elst 1
|
| City |
Wageningen |
| ZIP/Postal code |
6708 WD |
| Country |
Netherlands |
| |
|
| Platform ID |
GPL15007 |
| Series (1) |
| GSE125189 |
Granulosa cells of ovarian antral follicles exhibit distinct follicle size-related processes |
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