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Summary
This data set was generated as part of the RGAP project funded by NIAAA (R24 AA013162; MPI – Tabakoff, Hoffman, Saba) at the University of Colorado Anschutz Medical Campus. The processing of the RNA-Seq data was also supported by the NIDA Center of Excellence in Omics, Systems Genetics, and the Addictome (P30 DA044223; MPI – Williams, Saba). These data represent the RNA expression levels of Ensembl genes in brain. Ribosomal RNA depleted total RNA was used to generate paired end libraries. Levels were estimated in 45 (version 5) strains of the Hybrid Rat Diversity Panel (HRDP) including 30 HXB/BXH recombinant inbred strain and 15 classic inbred strains. Two to three biological replicates per strain were included in the strain-level means. Male rats were used for all analyses.
Experiment design
Predisposition database. RNA expression levels were measured on rats that had not been exposed to any type of intervention (e.g., alcohol, drugs, stress).
About cases
For this data set, a subset of the Hybrid Rat Diversity Panel (HRDP) was used that included 30 HXB/BXH recombinant inbred strains and 15 classic inbred strains for a total of 45 inbred strains. Male rats at the age of 90 days were used for this study. The animals from the 30 RI strains and the 2 progenitor strains of the HXB/BXH RI panel (SHR/OlaIpcv and BN-Lx/Cub) were bred and maintained at the Institute of Physiology of the Czech Academy of Sciences, Prague, Czech Republic. The 11 classic inbred strains were purchased from US Vendors (either Charles River or Envigo) and shipped to the University of Colorado Anschutz Medical Campus where they were acclimated prior to sacrifice. The remaining 2 classic inbred strains are the progenitor strains of the LEXF/FXLE RI panel and were bred and maintained at the National BioResource Project for the Rat in Japan. In Colorado, rats were sacrificed by rapid CO2 exposure. In the Czech Republic, rats were sacrificed by cervical dislocation. All experiments involving the harvesting of whole brain of the HXB/BXH RI panel and two progenitor strains were performed in accordance with the Animal Protection Law of the Czech Republic and were approved by the Ethics Committee of the Institute of Physiology, Czech Academy of Sciences, Prague. All experiments involving the harvesting of whole brain of the 11 classic inbred strains maintained at the University of Colorado Anschutz Medical Campus were approved by the Institutional Animal Care and Use Committee of the University of Colorado Anschutz Medical Campus and were performed in accordance with the guidelines in the NIH Guide for the Care and Use of Laboratory Animals. Animal experiments involving the F344/Stm and LE/Stm strains were conducted in accordance with the Fundamental Guidelines for Proper Conduct of Animal Experiment and Related Activities in Academic Research Institutions under the jurisdiction of the Ministry of Education, Culture, Sports, Science and Technology of Japan, and were approved by the Committee on Animal Experimentation at Kyoto University, Japan.
About tissue
Upon sacrifice, brains (including cerebellum and brain stem) were rapidly removed, sectioned sagittally into two hemispheres, and stored in liquid nitrogen. Each hemisphere was stored separately at -80⁰C until used for RNA extraction. Tissues from the Czech Republic and Japan were shipped on dry ice to the University of Colorado Anschutz Medical Campus for RNA extraction and cDNA library preparation
About platform
Total RNA was isolated from 3 biological replicates of each of the HRDP v5 strains using QIAzol (Qiagen, Valencia, CA, USA).The RNAeasy Plus Universal Midi Kit (Qiagen) was used to separate long (>200 nt) and short (<200 nt, miRNA-enriched) fractions. The long RNA fraction was purified using the RNeasy Mini Kit (Qiagen). Four microliters of a 1:100 dilution of either ERCC Spike‐In Mix 1 or Mix 2 (Thermo Fisher Scientific, Wilmington, DE) was added to each extracted RNA sample. Sequencing libraries for the long RNA fraction were constructed using the Illumina TruSeq Stranded Total RNA Sample Preparation Kit with Ribo-Zero ribosomal RNA reduction chemistry (Illumina) in accordance with the manufacturer’s instructions. An Agilent Technologies Bioanalyzer 2100 was utilized to assess sequencing library quality. A loading control was included in each sequencing batch. The loading control was a library generated from an SHR animal. Samples were sequenced (depending on batch either 2X100 or 2x150 paired end reads) on an Ilumina sequencer.
About data processing
Prior to alignment, reads were demultiplexed and read fragments were trimmed for adaptors and for quality using Cutadapt (version 1.9.1;(Martin, 2011)). Reads were eliminated if the trimmed length of either read fragment was <20 nucleotides. Next, bowtie2(v 2.2.6) (4) was used to align reads to ribosomal RNA, and the unmapped reads were retained. These will be referred to as “cleaned” reads. The RNA-Seq Expectation Maximization (RSEM v1.2.31) algorithm (Li and Dewey 2011) was used to estimate the number of aligned reads for individual Ensembl transcripts (version 96). The number of reads aligned to each Ensembl gene was calculated as the sum of the number of reads aligned to individual Ensembl transcripts annotated to that gene.
Prior to normalization and transformation, a detection above background filter was applied where we required a gene to have at least 2/3 of the samples have 1 count or more to be considered expressed above background. All genes that did not pass this detection above background criteria were removed. Count values were normalized using RUVg (PMID: 25150836) and transformed using the regularized log function (PMID: 25516281).
Contributors
Boris Tabakoff, PhD
University of Colorado Anschutz Medical Campus
Grant Support: NIAAA R24 AA013162
Paula Hoffman, PhD
University of Colorado Anschutz Medical Campus
Grant Support: NIAAA R24 AA013162
Laura Saba, PhD
University of Colorado Anschutz Medical Campus
Grant Support: NIAAA R24 AA013162 and NIDA P30 DA044223
Michal Pravenec PhD
The Czech Academy of Sciences
Grant Support: Academic premium of the Czech Academy of Sciences (AP1502)
Masahide Asano, PhD
Institute of Laboratory Animals, Graduate School of Medicine, Kyoto University
Grant Support: The National BioResource Project-Rat (Grant Number: 964706, 19 km 0210167j0001)
Melinda Dwinell, PhD
Medical College of Wisconsin
Grant Support: R24 OD024617
Citation
Tabakoff B., Smith H., Vanderlinden L.A., Hoffman P.L., Saba L.M. (2019) Networking in Biology: The Hybrid Rat Diversity Panel. In: Hayman G., Smith J., Dwinell M., Shimoyama M. (eds) Rat Genomics. Methods in Molecular Biology, vol 2018. Humana, New York, NY
Acknowledgment
Data were generated with funds provided by NIAAA, NIDA, the Banbury Fund, Czech Academy of Sciences, and The National BioResource Project-Rat. Dr. Michal Pravenec of the Czech Academy of Sciences kindly provided the tissues from the HXB/BXH recombinant inbred panel. Dr. Masahide Asano of the University of Kyoto kindly provided tissues from the F344/Stm and LE/Stm strains. Dr. Melinda Dwinell will be providing tissues for many of the remaining HRDP strains. We would like to thank Spencer Mahaffey, Jennifer Mahaffey, Yinni Yu, Seija Tililanen, Lauren Vanderlinden, and Harry Smith for help with extracting RNA, generating libraries, and processing data. We would also like to acknowledge the support of UNLV National Supercomputing Institute (UNLV NSI) by providing access to supercomputing resources to support analysis of sequencing data.