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Sample GSM2644709 Query DataSets for GSM2644709
Status Public on Jun 01, 2017
Title 2080120_TimePoint5_WholeBlood
Sample type SRA
 
Source name whole blood
Organisms Macaca mulatta; Plasmodium cynomolgi strain B
Characteristics time point: 5
gender: Male
mahpic non human primate individual id: RMe14
infected with: Plasmodium cynomolgi strain B
Treatment protocol During the 100-day experiment, artemether was delivered on days 27 and 53 (corresponding to time points 3 and 4), for blood-stage treatment. Chloroquine and primaquine were administered at the end of the study to treat liver- and blood-stages.
Growth protocol Animals approved for use were moved into experimental housing 10 days prior to the start of the experiment.
Extracted molecule total RNA
Extraction protocol Whole blood (3 ml) was collected in Tempus tubes (Applied Biosystems) that also preserve mRNA; these samples include erythrocytes, platelets and granulocytes in addition to mononuclear lymphocytes. RNA was extracted using Tempus-Spin RNA isolation kits. The quality of all RNA samples was confirmed using a Bioanalyzer, with an RNA Integrity Number (RIN) greater than 8 recorded for all samples.
Approximately 1 μg of total RNA per sample was converted to double-stranded cDNA using poly-A beads to enrich for mRNA, and Illumina TruSeq Stranded mRNA Sample Prep kits to generate strand-specific libraries. As a quality control, 96 spike-in RNAs of known concentration and GC proportions (ERCC Spike-In Control, Life Technologies) were added to constitute approximately 1% of the total RNA for each library. Adapters were ligated to facilitate 3-plex sequencing on an Illumina HiSeq2000 at the Yerkes Genomics Core, aiming for 80 million paired-end 100 base pair (bp) reads per library.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina HiSeq 2000
 
Description E04M99FGMmCyDaBM_Cynomolgi-Genes-LibSizeNormalized-Results_MULTIPL_GEO.xlsx
E04M99FGMmCyDaBM_Mulatta-Genes-LibSizeNormalized-Results_MULTIPL_GEO.xlsx
Data processing Bases were called with Illumina RTA (Real-Time Analysis, v1.13.48) with default parameters. FASTQC (v0.10.1) was used to assess data quality, but the data were not filtered at this stage.
To quantify gene expression, RNA-Seq reads were mapped to the listed assembly and annotation using Tophat2 (Trapnell et al, 2013). Default options were used with the exception that the command --library-type fr-secondstrand was used since reads were generated using a stranded library preparation method from Illumina. This allowed differentiation between sense and antisense transcripts. Only reads that map to a single location in the genome were included, to ensure high-confidence mapping.
Several quality control steps were used to verify the reliability of the data: linear correlation of estimated abundance of ERCC spike-in controls with known concentration; confirmation of 99.9% strand-specificity of the controls; less than 0.1% control fusion transcripts; and absence of 3’ bias in the controls was confirmed with RSeqC software (https://code.google.com/p/rseqc). Transcript abundance levels were inferred using HTSeq v0.5.4 (http://www-huber.embl.de/users/anders/HTSeq/doc/). HTSeq takes the short-read mapping file (bam) from tophat2 and the gene annotation file which contains the locations of all annotated genes. Since some libraries were sequenced more deeply than others, the libraries were normalized before determining differential gene expression using the gene level expression files with the default parameters of DESeq v1.10.1 (http://www.bioconductor.org/packages/release/bioc/html/DESeq.html).
Genome_build: RNA-Seq reads were mapped to both a host and parasite genome. Host: An early version of a new  assembly (as of 5/2014) of the rhesus macaque (MacaM assembly, v4.0, created by Aleksey Zimin at the University of Maryland, Rob Norgren at the University of Nebraska Medical Center and their colleagues. The MacaM assembly has been deposited in GenBank under accession PRJNA214746 ID: 214746. Parasite: PlasmoDB version 9.3 of the Plasmodium cynomolgi B strain genome assembly was used. The assembly has been deposited in GenBank under the accession PRJDA49901 ID: 49901.
Supplementary_files_format_and_content: Excel files contain normalized transcript abundances at the gene level, for each individual. Abundances are further classified by experimental Time Point (1-7), and Specimen Type (Whole Blood). One sample is from a transfusion donor, animal ID = CF97.
Supplementary_files_format_and_content: Gene Data Column headers are defined as follows: 'gene_name': Identifiers of all Genes in the reference annotation. 'gene_symbol': Symbols of all Genes in the reference annotation. 'Sample ID / Raw File 1 / Raw File 2 /Normalized Read Counts': Sample ID, Raw sequence file names for the sample, and Library size normalized and Log2 transformed read counts of all genes, from the Specimen Type, Individual ID, and Time Point listed directly below. Notes: There will be one read count column per specimen per NHP (Non-Human-Primate) per Time Point. So, 1 * 5 * 7 = 35 columns with read counts. Note that one NHP was euthanized during the experiment, so the total number of samples is 31. 0 as value for read counts of some genes Overlapping genes (sharing exons) were "collapsed" into a single gene for the purposes of RNA-Seq read assignments. So, the read count at such a locus is representative of the cumulative expression of all the genes at that locus. BUT, the entire read count is assigned to only one of the genes and the others in the locus are assigned 0.
 
Submission date May 31, 2017
Last update date May 15, 2019
Contact name Mary Galinski
Organization name Emory University
Department Vaccine Center at Yerkes
Lab Galinski Lab
Street address 954 Gatewood Road
City Atlanta
State/province GA
ZIP/Postal code 30329
Country USA
 
Platform ID GPL25692
Series (2)
GSE94274 An Integrated Approach to Understanding Host-Pathogen Interactions
GSE99486 Malaria Host Pathogen Interaction Center Experiment 04: Host and parasite gene transcript abundances, from whole blood, of Macaca mulatta infected Plasmodium cynomolgi treated with artemether over 7 time points in a 100 day study
Relations
BioSample SAMN07180351
SRA SRX2872567

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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