METHODS
Expression profiling and data analysis
Quality of all RNA samples was determined using Agilent 2100 Bioanlyzer (S28/S18>1.8, and RIN>9) and
500ng of high quality RNAs were used to synthesize biotin labeled cRNA using MessageAmp IITM RNA
Amplification kit (Applied Biosystems, Cat# 1751). Quality of cRNA amplification was verified using Agilent
2100 Bioanlyzer, and 1.5ug cRNA was heated to 65°C for 5 min, and loaded onto the Illumina whole genome
mouse platform (V 1.1) BeadChip. The BeadChips were sealed in a hybridization chamber and placed in an
oven at 58°C with a rocker for 20 hrs. After the hybridization, the BeadChips were washed, stained, and
scanned on the Illumina BeadArray Reader. Scanned files were initially processed using the LUMI package
(www.bioconductor.org/packages/bioc/html/lumi.html) in R (cran.r-project.org). Probes were filtered for
detection (p<.0005), subjected to a variance stabilization transformation, and quantile normalization procedure.
Quality control analysis revealed that one array (i.e. # 4051964056_D) was a significant outlier with respect to
the other arrays and was removed from further analysis. All 46,643 array probes were remapped against the
Refseq mouse transcriptome (NCBI 37/mm9) using the Seqmap program (Jiang and Wong, 2008). Only
probes that were a perfect match to Refseq transcripts were analyzed further. A total of 23,034 probes were
successfully mapped to the Refseq transcriptome. A total of 8,293 of these probes passed our expression
criterion and were then pre-filtered as described in (Bourgon et al, 2010) using the following two step
procedure: First, genes were log2 transformed and variances of expression irrespective of experimental group
were calculated. Second, genes with variances greater than the median were retained for further analysis,
leaving 4,146 probes. These probes were analyzed using the MAANOVA package (cran.rproject.org/web/packages/maanova/index.html) in R. Data software, and fitted to a mixed-effect model with
beadchip as a random effect and group (control versus BDS) as a fixed effect. For this dataset, two beadchips
were used, one with 5 samples/arrays and the other with 6 samples/arrays. P values were calculated using
permutation tests and a total of 1000 permutations were computed. P values based on the "Fs" statistic (Cui et
al, 2005) and then subjected to multiple comparison correction (Benjamini and Hochberg, 2005). Probes with
false discovery rate (FDR)-corrected P values (Q values) less than 0.1 were considered to be significant (see
Table S1 in the supplemental material section).
FIGURE LEGENDS FOR SUPPLEMENTAL MATERIAL
Table S1. This table provides a list of 192 genes with nominal value of p< 0.05, presented in an ascending
order based on their false discovery rate (FDR) corrected values. Genes that passed the FDR of Q> 0.1 are
highlighted. The columns provide information regarding: probe ID, gene symbol, accession number, fold
change (BDS/control), levels of gene expression in the BDS and control groups, Permutated P value, FDR
value, and function (shown only for the genes that passed FDR of Q> 0.1). Values presented in the fold
change (BDS/control), and expression level columns are based on log 2 transformed data that were used in
the statistical analysis.
Figure S1. BDS is associated with increased postnatal maternal care and prolonged elevation of corticosterone
levels in PND14 pups. Levels of maternal care in BDS (n=13 litters) and control (n=12 litters) groups were
monitored for the first postnatal week and are summarized for LG (a), ABN (b), passively nursing (c), and no
contact with the pups (d). Pups were exposed to daily BDS (n=14) or control condition (n=8) and levels of
plasma corticosterone were assessed in PND14 pups at 50 min (n=6, 3 independent litters) or 120 min (n=8, 4
independent litters) after BDS exposure. Note that at 50 and 120 min time points the pups were reunited with
their dam and back in their home cage for roughly 35 and 105 min respectively. Abbreviations: ABN-arched
back nursing, LG-licking and grooming. Results in a-d are expressed as % frequencies averaged over PND 1-7
and s.e.m, (a-d) unpaired Student t test, (e) one way ANOVA followed by Tukey-HSD post-hoc analysis
compared to control condition. #p= 0.059, *p< 0.05,***p< 0.0005.
Figure S2. Representative autoradiographs after in-situ hybridization using anti-sense probe (top) and sense
probe (bottom) for LBP. Note that equal amounts of anti-sense and sense probes were used and that exposure
time and image processing were identical for both probes.
Figure S3. (a) Fifty microgram of hippocampal lysate from PND14 LBP ko and wildtype littermates were
separated on 10% SDS-PAGE, transferred to a nitrocellulose membrane, and probed using goat anti LBP
antibodies (1:1000, Santa Cruz, Cat # sc-14668) and Rabbit anti beta tubulin (1:20,000, abcam cat# ab6046).
Membrane was incubated with the fluorescently labeled secondary antibodies (Li-Cor) and scanned on an
Odyssey infrared imaging system ( Li-Cor Biosciences). (b-c) PND 21 LBP ko and littermate wildtype control
pups were perfused and hippocampal slices were stained with goat anti LBP antibodies (1:100, Santa Cruz,
Cat # sc-14668) and detected with DyLightTM 488 donkey anti-goat (1:250, Jackson ImmunoResearch). Low
magnification confocal pictures showed a punctate pattern of staining throughout the hippocampus in wildtype
animal (b) but not in LBP ko littermate (c). Scale bars are 10um.
REFERENCES
Benjamini Y, Hochberg Y (2005). Controlling the false discovery rate: A practical and powerful approach to
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Jiang H, Wong WH (2008). SeqMap: mapping massive amount of oligonucleotides to the genome.
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