|
| Status |
Public on Oct 20, 2019 |
| Title |
unmatched reads from pigtailed macaque donors AG07923 and AG08490 |
| Sample type |
SRA |
| |
|
| Source name |
Dermal fibroblasts
|
| Organism |
Macaca nemestrina |
| Characteristics |
cell type: primary dermal fibroblasts
treatment: 24 h transfection 0.05 ug/cm2 poly(I:C) and 24 h mock transfected
passage: AG07923 (p10), AG08490 (p5)
|
| Treatment protocol |
Cells were seeded the night before transfection with 200,000 cells/well in a 6 well format so that at the time of transfection they were at least 80% confluent. Each well received 0.5 ug high molecular weight poly(I:C) using using X-tremegene HP DNA transfection reagent in Opti-MEM.
|
| Growth protocol |
All cells were grown under standard conditions in Dulbecco’s modified Eagle medium containing 10% fetal bovine serum (FBS) and 1% v/v penicillin/streptomycin.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA extracted using Qiagen Rneasy Mini Kit per manufacturer's directions.
The integrity of total RNA from the primate samples were assessed on Bioanalyzer 2100 using RNA 6000 Nano chip (Agilent Technologies, CA), only samples with RNA Integrity Number (RIN) greater than 9.0 were used for RNA-seq. One microgram of total RNA from each sample was subject to poly-A containing RNA enrichment by oligo-dT bead and converted to cDNA then sequencing library using PrepX RNA-seq library kit on the automated Apollo 324TM NGS Library Prep System (Wafergen Biosystems, CA) according to the manufacturer's protocol. Different DNA barcodes were added to each library. The libraries were examined on Agilent Bioanalyzer DNA High Sensitivity chips for size distribution, quantified by Qubit fluorometer (Invitrogen, CA). The RNA-seq libraries were pooled at equal molar amount and sequenced on Illumina HiSeq 2500 Rapid flowcells as single-end 75nt reads following the standard protocol. Raw sequencing reads were filtered by Illumina HiSeq Control Software and only the Pass-Filter (PF) reads were used for further analysis.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
Unmatched reads from multiple samples either mock transfected or transfected in triplicate in a 6 well format for 24 h with 0.5 ug poly(I:C) (~0.05 ug/cm2) per well
|
| Data processing |
Samples sequenced on SN387 with HiSeq Rapid SBS Kit v2. Image analysis and base-calling was performed with RTA version 1.18.64.0.
Barcode splitting performed in Galaxy with 1 mismatch allowed unless otherwise stated in the Samples, column Special notes. Short reads were aligned to the human or the originating species’ genomes using RNA STAR (Galaxy version 2.6.0b-1) with default parameters. Counts were generated using featureCounts with default settings (Galaxy version 1.6.3+galaxy2), downloaded, and read into R version 3.5.2 (2018-12-20) using scripts run in RStudio version 1.1.463.
htseq-count Gene counts analyzed further with DESeq2 (version 1.22.2) (Love MI, Huber W and Anders S (2014) “Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2.” Genome Biology, 15, pp. 550. doi: 10.1186/s13059-014-0550-8) in RStudio (R version 3.3.3.), using two factors (donor + treatment) as the experimental design.
genome build
Each species was aligned to human and its specific reference genome except for human (which was only aligned to the human genome) and mouse (which was only aligned to the mouse genome).
Species
Orangutan: ppyg2 (Ensembl 93)
Gorilla: gorGor4 (Ensembl 95)
Pigtailed macaque : Mnem_1.0 (Ensembl 95)
Rhesus macaque: Mmul_8_0_1 (Ensembl 95)
Bonobo: panpan1.1 (Ensembl 95)
Chimpanzee: Pan_tro_3.0 (Ensembl 95)
Olive baboon: papAnu2 (Ensembl 95)
Squirrel monkey: SaiBol1.0 (Ensembl 95)
Human: GRCh38 (Ensembl 96)
Mouse: GRCm38 (Ensembl 85)
processed data files format and content: all tab-delimited text files of htseq-count "Gene counts" :
|
| |
|
| Submission date |
Oct 19, 2017 |
| Last update date |
Oct 20, 2019 |
| Contact name |
Alexander Ploss |
| E-mail(s) |
aploss@princeton.edu
|
| Phone |
609-258-7128
|
| Organization name |
Princeton University
|
| Department |
Molecular Biology
|
| Lab |
110 Lewis Thomas Laboratory
|
| Street address |
Washington Road
|
| City |
Princeton |
| State/province |
NJ |
| ZIP/Postal code |
08544 |
| Country |
USA |
| |
|
| Platform ID |
GPL19597 |
| Series (1) |
| GSE105160 |
RNASeq of mouse, human, and non-human primate primary dermal fibroblasts to poly(I:C) transfection |
|
| Relations |
| BioSample |
SAMN07814469 |
| SRA |
SRX3300231 |
