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| Status |
Public on Feb 22, 2019 |
| Title |
9993000005_RingStage(B+1+)_Cultured Cells |
| Sample type |
SRA |
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| Source name |
Cultured Cells
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| Organisms |
Plasmodium knowlesi; Macaca mulatta |
| Characteristics |
time point: RingStage
gender: Male
non human primate individual id: RDv7
infected with: Plasmodium knowlesi
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| Growth protocol |
Animals approved for use were already in experimental housing. Sample dates range from 9/14/07 to 6/14/17. All Non Human Primates used in this experiment were naive for the particular clone of P. knowlesi used to generate the time course. Macaca mulatta were inoculated with a particular parasite clone and allowed to reach a targeted peak parasitemia of 5% - 10% proportion of infected RBCs. Whole blood was collected at the early ring stage while subjects were under anesthesia, and an ex vivo culture was established using standard protocols, which include but are not limited to the processing of the whole blood by removing platelets and white blood cells. Samples were collected starting at 1.5-2 hours after establishing cultures, followed by collections every 4 hours for a 24 hour period.
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| Extracted molecule |
total RNA |
| Extraction protocol |
(Ex_vivo_prep_of_RBCs.pdf SOP) 1.RBCs are obtained from an infected rhesus monkey. 1.a. 10 ml of blood/kg of weight are drawn 1.b. Sample is collected in heparin tubes 1.c. Pool individuals tubes in a 50ml-tube to obtain total volume 2. Platelets are removed by the addition of adenosine diphosphate (SIGMA A-4386; Stock: 40 mg/ml prepared in PBS) 2.a. Add 0.5 ml per 10 ml of blood 2.b. Incubate 5 min at room temperature 3. Preparation of columns: 3.a. Glasperlen Glass bead column 3.a.i. Use a ___ ml syringe 3.a.ii. Bed volume of glass beads: 1/2 volume of blood 3.a.iii. Pre-wet beads with Hanks Solution (HBSS) 3.b. Cf11 Column: 3.b.i. Use __ml syringe 3.b.ii. Volume of cellulose: 2X of total volumne 3.b.iii. Mix cellulose with HBSS, 50ml of cellulose + 100ml of HBSS gives aprox. 37ml of bed 3.b.iv. Pour into syringe at once and don't allow to drip until ready to start 3.c. Add blood carefully into glass bead column 3.d. Push with the plug gently 3.e. Wash with one column volume of HBSS 3.f. Remove the plug from CF11 column 3.g. Drip blood through CF11 column 3.h. Allow blood to flow completely into cellulose matrix 3.i. Wash CF11 column with one column volume 3.j. Collect fraction in 50ml tubes 3.k. Centrifuge 2000 rpm for 5 min (RPMI recipe.pdf SOP) 1. RPMI - 1640 Complete medium or McCoys 1.a. 1 package RPMI 1640 powder or McCoys powder 1.b. 0.5 ml gentamicin (50 mg/ml) 1.c. 7.2 gm HEPES 1.d. 2 ml (25mg/ml) Hypoxanthine in 1 M NaOH 1.e. 2 gm Glucose (Dextrose) for RPMI or 1 gm for McCoys 2. Dissolve RPMI-1640 in 500 ml Gibco distilled H2O in 1 liter volumetric flask 3. Add various ingredients and mix 4. Adjust pH to 7.2 with 1M NaOH 5. Bring to total volume 1000ml with H2O 6. Filter sterilization 7. Add 48ml serum (10%) and 16ml bicarbonate (0.25%) to 432ml RPMI 8. Filter sterilization (Cell_Culture) 1. Processed RBCs are put into standard culture medium in culture flasks with plug seal caps. 2. Culture flasks are then gassed with blood gas mix 3. Incubate cultures at 37 degrees celsius (Total_RNA_Extraction_from_Parasite_Cultures.docx SOP) 1. Spin down the cultures at 700 g for 5 min. Aspirate off the supernatant. 2. Add 5 volumes of pre-warmed TRIzol LS (37°C) and shake vigorously for 1 min. 3. Incubate at 37°C for 5 min. 4. Keep the samples on ice. For each 5 mL of TRIzol LS that was used in step 2, add 1 mL of chloroform and vortex/shake for 1 min and transfer to a Teflon tube. 5. Centrifuge at 12000 g for 30 min at 4°C. 6. Carefully transfer the upper aqueous layer to a fresh polypropylene tube and add 0.8 volume of pre-chilled isopropanol to precipitate the RNA. Mix carefully by inverting. 7. Mix by inverting and centrifuge at 12000 g for 30 min and 4°C. Carefully aspirate off the supernatant. 8. Allow the pellet to air-dry on ice for 5 min and add 30-100 μL of RNase-free non-DEPC treated water. 9. Heat tubes at 60°C for 10 min and then place on ice. 10. Clean up RNA with the Qiagen RNeasy kit, with the optional DNAse digest on the column. 11. Measure the RNA conc and absorbance on Nanodrop. 12. Store aliquots for northern blots (30 ug total RNA in ETOH+NaAc), PCR (2 ug in water), and RNAseq (5 ug in water). 13. Total RNA (5 µg) of each sample (except sample 9993000009, see sample section for more information) were used for sequencing. 14. The quality of all RNA samples was then 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, 92 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 multiplexed sequencing on an Illumina HiSeq3000 at the Yerkes Genomics Core, aiming for 100 million paired-end 100 base pair (bp) reads per library.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
Bases were called with Illumina RTA (Real-Time Analysis, v2.7.7) with default parameters. Illumina bcl2fastq v2.17.1.14 was used for demultiplexing. FASTQC (v0.10.1) was used to assess data quality, but the data were not filtered at this stage.
Reads were mapped to a composite reference assembly consisting of host, parasite, and ERCC control references with STAR (v2.5.2b) with default alignment parameters. Note all values for ERCC abundances will be zero as no spike ins were included in sequenced samples. See readme for details.
Abundance estimation of raw read counts per transcript was done internally with STAR using the algorithm of htseq-count.
Normalized expression (normalized read counts) was performed with DESeq2 (v1.10.1)
Genome_build: RNA-Seq reads were mapped to 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 BioProject accession PRJNA214746. Parasite: Plasmodium knowlesi Malayan Strain Pk1 (A+) genome assembly was used. The assembly has been deposited in GenBank under the BioProject accession PRJNA377737.
Supplementary_files_format_and_content: Excel files contain either normalized transcript abundances or raw counts at the gene level, for each individual. Abundances and raw counts are further classified by experimental Time Point (RingStage), and Specimen Type (Cultured Cells).
Supplementary_files_format_and_content: Host, Parasite, and ERCC Raw Read Counts Column headers are defined as follows: 'Gene ID': Identifiers of all Genes in the annotation. 'Gene Symbol': Symbols of all Genes in the annotation. 'Raw File List / Sample Identifier / Abundances': Samples were sequenced across multiple lanes and all fastq files are listed for each sample. Rawread counts of all genes, from the raw files listed in the column header. The raw file names and sample identifer both contain information regarding Specimen Type, NHP ID, and Time Point. Notes: There are 9 columns representing 9 subjects. There is an read count entry per sample for every gene that appears in the annotation. For genes where there was no detection of expression by reads that mapped to their loci, the raw count is 0.
Supplementary_files_format_and_content: Host DESeq2Normalized Counts Column headers are defined as follows: 'Gene ID': Identifiers of all Genes in the annotation. 'Gene Symbol': Symbols of all Genes in the annotation. 'Raw File List / Sample Identifier / Abundances': Samples were sequenced across multiple lanes and all fastq files are listed for each sample. DESeq2 normalized read counts of all genes, from the raw files listed in the column header. The raw file names and sample identifer both contain information regarding Specimen Type, NHP ID, and Time Point. Notes: There are 9 columns representing 9 subjects. There is an read count entry per sample for every gene that appears in the annotation. For genes where there was no detection of expression by reads that mapped to their loci, the raw count is 0.
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| Submission date |
Feb 21, 2018 |
| Last update date |
Feb 25, 2019 |
| Contact name |
Mary Galinski |
| Organization name |
Emory University
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| Department |
Vaccine Center at Yerkes
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| Lab |
Galinski Lab
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| Street address |
954 Gatewood Road
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| City |
Atlanta |
| State/province |
GA |
| ZIP/Postal code |
30329 |
| Country |
USA |
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| Platform ID |
GPL25695 |
| Series (2) |
| GSE94274 |
An Integrated Approach to Understanding Host-Pathogen Interactions |
| GSE110970 |
Malaria Host Pathogen Interaction Center Experiment S01: Host and parasite gene transcript abundances, from cultured cells, of Macaca mulatta infected with Plasmodium knowlesi Pk1(A+), Pk1(B+)1+, Pk1(C+) ex vivo time course. |
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| Relations |
| BioSample |
SAMN08580600 |
| SRA |
SRX3733704 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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