All cell lines and tissues were sourced from 8-10 week old male C57Bl/6 mice, with the exception of female-specific organs, which were sourced from female mice. All procedures were carried out in accordance with local guidelines for animal research. For female tissues, material was pooled from three females, and for each female, on average four embryos resulting in four umbilical cords and placentas were obtained. For other tissues, material was derived from a pool of three males. Biological replicates were defined as independent RNA preparations from independent pools of mice. Technical replicates were defined as independent amplifications from the sample RNA sample.
Extracted molecule
total RNA
Extraction protocol
RNA extraction from mammalian cells or tissues was performed using RNeasy kits (Qiagen, Valencia, CA) or a standard Trizol protocol. If tissue amounts were more than 50 mg per mouse when dissected, tissues were pulverized while frozen. RNA was prepared separately for every mouse in order to identify samples with potentially degraded RNA. Trizol-extracted RNA was purified with a Qiagen RNeasy column. For bone marrow macrophages, osteoblasts, and osteoclasts, contaminating genomic DNA was removed during the RNeasy cleanup using DNaseI (Qiagen, Valencia, CA). The integrity and concentration of RNA was determined via microfluidic analysis on a bio-analyser (Agilent Technologies, Palo Alto, CA) or by analysis on a BioRad Experion. Pooling occurred at the RNA level.
Label
biotin
Label protocol
For samples containing more than two µg total RNA available after pooling, standard Affymetrix single amplification was performed using two µg total RNA. For pooled samples containing less than two µg total RNA, 100 ng total RNA (or 50 ng for thymocyte_SP_CD8+) was used in a standard Affymetrix double amplification protocol.
