normalization data transformation protocol
Images were quantified using Agilent Feature Extraction software version 10.7.3.1 ID_REF = VALUE = Log2 normalized signal intensity.
array scanning protocol
Microarray slides were scanned in an Agilent Technologies G2505C Microarray Scanner at 5 micron resolution.
Hybridizations were performed for 17 hrs, according to the manufacturer’s protocol.
RNA was labeled with Cy3-CTP using the Agilent’s Low Input Quick Amp Labeling Kit following the manufacturers protocol. Labeled cRNA was assessed using the Nanodrop ND-1000 UV-VIS Spectrophotometer version 3.8.0. (Nanodrop Technologies, Inc., Wilmington DE), and equal amounts of Cy3 labeled target were hybridized to Agilent SurePrint G3 Mouse GE 8×60K microarray.
nucleic acid extraction protocol
Total RNA was isolated using Isogen (Nippon Gene Co., Ltd., Tokyo, Japan) according to the manufacturer's protocol and suspended in pure water. The RNA quantity was determined by spectrophotometry measurement of OD260/280 (ratio > 1.8) in a BioPhotometer plus (Eppendorf, Hamburg, Germany). After purification of RNA by ethanol precipitation and an RNeasy Micro Kit (Qiagen, Hilden, Germany), the RNA integrity was evaluated by capillary electrophoresis using a Bioanalyzer 2100 (Agilent Technologies, Inc., Santa Clara, CA, USA).
Pregnant C57BL/6J mice, weighing approximately 30 g (Figure S1A) at gestational day 14, were purchased from CLEA Japan, Inc. (Tokyo, Japan) and used for experiments. All animals were acclimated to our animal room (The Center for Environmental Health Science for the Next Generation, Research Institute for Science and Technology, Tokyo University of Science). They had free access to water and standard animal food and were exposed to a 12-hour light/dark cycle (lights on between 8:00 and 20:00), a temperature of 22±1 °C, and a humidity-controlled environment (50±5%).
sample treatment protocol
Upon arrival to the colony, half of the pregnant dams were assigned to the standard cage environment (C) and the other half to environmental enrichment (EE). Standard laboratory cage consisted of a common housing cage for mice (30 × 20 × 12.5 cm: 7500 cm3). Environmental enrichment consisted of a larger (40 × 25 × 19 cm: 19,000 cm3) cage containing a running wheel, small house, wood blocks, and plastic tubing that were moved to different locations within each cage every 2–3 days and were exchanged with new toys. One dam and her pups (n=8) were housed in either the standard laboratory cage or environmental enrichment throughout the perinatal period and until weaning. After weaning at postnatal day 27, the offspring mice were placed in a control chamber or DE inhalation chamber and were housed under the same conditions as during the perinatal period. The mice were exposed to DE for 8 hours/day (10:00–18:00) for 28 days (postnatal days 28−55) in the control or DE inhalation chamber at the Center for Environmental Health Science for the Next Generation (Research Institute for Science and Technology, Tokyo University of Science). Housing environment (standard cage environment [C] or environmental enrichment [EE]) during the perinatal period (gestational day 14–postnatal day 28) and chamber (control [C] and [DE]) established four experimental groups: C-C, C-DE, EE-C, and EE-DE (Figure 1). Necropsies were performed 1 day after the final exposure. The olfactory bulb was collected from each male mouse, frozen quickly in liquid nitrogen, and then stored at −80 °C until total RNA extraction. Lung tissues were also collected and immersed in 10% phosphate buffered formalin until use.