strain: WSB/EiJ dam x PWD/PhJ sire F2 tissue: testis
Treatment protocol
The left testis was flash-frozen in liquid nitrogen upon dissection and stored at -80°. Frozen testis samples were transferred to RNAlater-ICE buffer (Invitrogen, Grand Island, NY, USA), shipped to the Max Planck Institute in Plön and stored at -80° until processing.
Growth protocol
Pups were weaned into same-sex sibling groups at 21 days, and males were separated into individual cages at approx. 56 days. Males were killed for phenotyping at 70 (±5) days of age using carbon dioxide.
Extracted molecule
total RNA
Extraction protocol
We extracted RNA from testis using RNeasy kits (Qiagen, Hilden, Germany). RNA quality (RIN > 8) was verified using RNA 6000 Nano kits (Agilent) on a 2100 Bioanalyzer (Agilent).
Label
Cy3
Label protocol
We used single-color Quick-Amp Labeling Kits (Agilent) to amplify and label RNA. The yield (>2 μg) and specific activity (>9.0 pmol Cy3/μg cRNA) of labeling reactions was verified using a NanoDrop ND-1000 UV-VIS Spectrophotometer (NanoDrop, Wilmington, DE, USA).
Hybridization protocol
Hybridization was performed following the One-Color Microarray-Based Gene Expression Analysis (Quick Amp Labeling) Protocol from Agilent.
Scan protocol
Arrays were scanned using a High Resolution Microarray Scanner (Agilent) and raw images processed using Feature Extraction Software (Agilent). Raw images and the distribution of non-uniformity outliers were visually inspected for large spatial artifacts (e.g. caused by buffer leakage or dust particles). We used quality control metrics from Feature Extraction protocol GE1_QCMT_Dec08.
Description
testis
Data processing
We mapped the 41,174 non-control probe sequences from the Whole Mouse Genome Microarray to the mouse reference genome (NCBI37, downloaded March 2011) using BLAT (minScore = 55, default settings for all other options). We excluded probes with multiple perfect matches, more than nine matches, matches to non-coding/intergenic regions only, and matches to more than one gene. A total of 36,323 probes (covering 19,742 Entrez Genes) were retained. Preprocessing of microarray data was performed using the BioConductor package Agi4x44PreProcess [91]. We used the “half” setting, which sets intensities below 0.5 to 0.5 following background subtraction, and an offset value of 50. The background signal was computed in Feature Extraction. During preprocessing, we filtered probes based on flags from Feature Extraction. Probes with signal above background in at least 10% of samples were retained (settings: wellaboveBG=TRUE, isfound=TRUE, wellaboveNEG=TRUE). We used default settings for quality-control flags. Following preprocessing, we removed probes for which the 98th percentile was <2-fold greater than background. The final data set includes 24,675 probes.
