Supplementary Material Study Sample the Vervets Used in This
Total Page：16
File Type：pdf, Size：1020Kb
Supplementary Material Study sample The vervets used in this study are part of a pedigreed research colony that has included more than 2,000 monkeys since its founding. Briefly, the Vervet Research Colony (VRC) was established at UCLA during the 1970’s and 1980’s from 57 founder animals captured from wild populations on the adjacent Caribbean islands of St. Kitts and Nevis; Europeans brought the founders of these populations to the Caribbean from West Africa in the 17th Century 1. The breeding strategy of the VRC has emphasized the promotion of diversity, the preservation of the founding matrilines, and providing all females and most of the males an opportunity to breed. The colony design modeled natural vervet social groups to facilitate behavioral investigations in semi-natural conditions. Social groups were housed in large outdoor enclosures with adjacent indoor shelters. Each enclosure had chain link siding that provided visual access to the outside, with one or two large sitting platforms and numerous shelves, climbing structures and enrichments devices. The monkeys studied were members of 16 different social matrilineal groups, containing from 15 to 46 members per group. In 2008 the VRC was moved to Wake Forest School of Medicine’s Center for Comparative Medicine Research, however the samples for gene expression measurements in Dataset 1 (see below) and the MRI assessments used in this study occurred when the colony was at UCLA. Gene expression phenotypes Two sets of gene expression measurements were collected. Dataset 1 used RNA obtained from whole blood in 347 vervets, assayed by microarray (Illumina HumanRef-8 v2); Dataset 2 assayed gene expression by RNA-Seq, in RNA obtained from 58 animals, with seven tissues (adrenal, blood, Brodmann area 46 [BA46], caudate, fibroblast, hippocampus and pituitary) measured in each animal. Dataset 1: Microarray (blood) The microarray data set has been described in Jasinska et al. 2 and is available at NCBI at the BioProject PRJNA115831. Briefly, Total RNA from whole blood preserved in PaxGene RNA Blood tubes (PreAnalyticX) was extracted using PAXgene Blood RNA Kit (PreAnalyticX). The integrity of the extracted total RNA samples was assessed on the Agilent 2100 Bioanalyzer (Agilent Technologies) with the RNA 6000 Nano Assay Kit (Agilent Technologies), and sample concentrations were quantified with RiboGreen RNA (Invitrogen). For assessing transcript levels, we used the Illumina HumanRef-8 v2 chip. This chip provides genome-wide transcriptional coverage of well-characterized genes and splice variants. This chip uses 22,184 probes representing 18,189 unique human genes (or 20,424 unique transcripts) from Reference Sequence (RefSeq) database1, Release 17. The Illumina gene expression platform utilizes 50-mer gene-specific probes that provide both good selectivity and sensitivity 3. We expected that the long probes would also tolerate sequence incompatibilities between human probe sequence and vervet target transcripts and be more robust than shorter probes to possible allelo-specific differences in hybridization efficiency due to vervet-specific SNP variants occurring in probe-interaction sites 4. cDNA was synthesized and in vitro transcribed into biotinylated cRNA using the Illumina Totalprep RNA amplification kit, following the manufacturer’s instructions (Ambion). Labeled cRNA was hybridized to the HumanRef-8 version 2 (Illumina) gene expression bead-chip. A gene was called detectable by BeadStudio when the detection p-value was <0.01. The gene expression module of the BeadStudio software version 3.1 (Illumina) was used for initial data processing and background correction. Lumi software was also used to perform a variance-stabilizing transformation that takes advantage of the technical replicates available on every Illumina microarray (usually over 30 randomly distributed beads per probe), and subsequently performs robust spline normalization and quality control of gene expression measures 5. Probe filtering for eQTL analysis: The probes on the Illumina HumanRef-8 v2 microarray were originally developed for assaying gene expression in humans. We selected probes that were compatible with vervet genomic sequence based on the following criteria: contain no indels, up a total of five mismatches in a probe, with maximum of one mismatch in the 16 nt central portion of the probe). To prevent expression measurement-bias due to SNP interference with the hybridization process, we excluded probes targeting sequences with high-quality common SNPs identified in the VRC pedigree. For this filtering step, we used a set of 3.45 Mln high-quality SNPs segregating in the VRC with MAF>=10%. A total of 11,001 probes passed these filters (Supplementary Data 1). We then evaluated the detection levels of each probe in all animals, and retained for analysis 6,018 probes that were detected with Illumina p<0.05 in at least 5% of animals. Dataset 2: RNA-Seq (seven tissues) Below we describe sample collection and processing, and determination of expression counts for the RNA-Seq gene expression dataset. Tissue collection for RNA: Tissues were obtained from 60 vervets during experimental necropsies. The vervets represented a range of developmental stages from neonates (7 days), infants (90 days and one year), young juveniles (1.25, 1.5, 1.75, 2 years old), peri- adolescents or subadults (2.5, 3 years old) to adults (4+ years old) with 6 vervets (3 males and 3 females) from each developmental time point. Two animals (a 1.75 year old female and a 7 day old male) were excluded for the eQTL study as they did not have WGS data, leaving a total of 58 monkeys with RNA-Seq and whole genome sequencing (WGS) data for eQTL analysis. Two samples (from caudate and BA46) from individual 2008147 were excluded after the PCA analysis suggested a sample mix-up between tissues. Vervets were anesthetized with 10-15 mg/kg of ketamine administered intramuscularly followed by pentobarbital 100 mg/kg administered intravenously. After animals reached a deep plane of anesthesia, the abdominal and thoracic cavities were opened and the vasculature was perfused with normal saline chilled to 4 C administered via the left cardiac ventricle until perfusate escaping through a cut in the○ inferior vena cava became blood free. After exsanguination, the head was then disarticulated and the top of the cranium removed using Ronjeurs to chip off small bone pieces. Keeping the Ronjeurs superficial to the dura, bone was first removed at the base of the skull, proceeding anteriorly just above the ear canal and across the brow ridge to fully encircle the entire head. The dura was then cut with scissors, and the top of the skull was removed with Ronjeurs. Scissors were used to cut cranial nerves II-XII to free the brain, keeping the olfactory nerve and olfactory bulb together with the brain when possible. After removal, the brain was weighed, and then hemisected with a scalpel. The right hemisphere was preserved in 10% neutral buffered formalin and the left hemisphere was dissected. Brain tissues were generally collected within 60-70 minutes of removal of the brain from the skull. All brain samples were placed immediately in RNAlater and refrigerated for 24 hours. The RNAlater was then removed and the tissue frozen at -80 C or below. ○ For 14 necropsies performed after August 4, 2010, the necropsy protocol was modified as follows: 1) 100% oxygen was delivered to the animal by face mask starting before pentobarbital administration and continuing until initiation of saline perfusion; 2) the animal was placed on a bed of ice prior to pentobarbital administration (but after ketamine administration) to accelerate cooling of tissues; 3) dissection protocols were streamlined to minimize the time elapsed between pentobarbital administration and removal of the brain from the skull and to minimize the time elapsed to the completion of brain dissection. During necropsies of 30 vervets whose brain tissues were used in this study, a fresh frozen sample of occipital lobe was collected near the end of the dissection procedure to allow tissue pH to be measured. pH was measured in occipital lobe specimens as described by Harrison et al.6. pH ranged from 5.98 to 6.98 (mean pH 6.6, SD 0.2). These data were used to assess the potential effect of brain pH on gene expression PC patterns (see Possible Technical and Biological Covariates). BA46: The samples were collected from both banks of the principal sulcus, bluntly dissecting downward with forceps inserted at the upper margin of each bank to free tissue to the floor of the principal sulcus. The midpoint of the principal sulcus along its length was used as the posterior margin for the collected samples, which extended anteriorly for the length of the sulcus. The samples collected from the two sulcal banks were pooled at the time of dissection and processed as a single sample. Caudate: From the medial surface of the hemisected brain, forceps were inserted into the lateral ventricle and used to remove the overlying tissue, exposing the caudate. A coronally oriented cut was then made through the head of the caudate, and caudate tissue was pinched from the portion of the brain anterior to this cut using forceps. Hippocampus: To obtain the hippocampal sample, a coronally oriented cut was made with a scalpel through the occipital pole, passing through the head and body of the hippocampus. Blunt dissection with forceps was used to isolate the hippocampus from surrounding tissue, extending approximately 2-3 mm into the exposed tissue face. RNA collection: The following tissue preservation methods were used during the collection to maintain sample quality. Whole blood was drawn into PAXgene RNA Blood Tubes (PreAnalyticX) with reagent instantly stabilizing RNA profiles. Solid tissues were preserved in RNAlater reagent (Ambion) immediately after collection, protecting the integrity of RNA profiles. To establish fibroblast cultures, skin biopsies were collected from shaved and cleaned skin from an area of the middle of the thigh (outer side) using a skin punch, after ketamine anesthesia but prior to euthanasia.