|
| Status |
Public on Apr 08, 2020 |
| Title |
7_283-09_Day_0 |
| Sample type |
SRA |
| |
|
| Source name |
Natural Killer (NK) cells
|
| Organism |
Macaca mulatta |
| Characteristics |
cell type: Natural Killer (NK) cells
|
| Treatment protocol |
Cells were thawed quickly in complete R10 media and immediately stained using antibodies to identify NK cells.
|
| Growth protocol |
Frozen primary cells were used for this experiment. Cells were frozen in 90%FBS + 10% DMSO and stored in LN2 vapor phase
|
| Extracted molecule |
total RNA |
| Extraction protocol |
NK cells were sorted directly into cold (4ºC) RPMI. The sorted NK cells were pelleted (500 x g, for 10 minutes) and lysed by vortexing for 1 minute in cold supplemented RLT buffer (RLT + β-MeOH) using the following ratio: 50µL cells in R10 and 350µL RLT buffer, 4ºC. Samples were then immediately frozen at -80ºC. RNA was extracted from these samples using the RNeasy Micro kit (Qiagen) with on-column DNase digestion. RNA quality was assessed using an Agilent Bioanalyzer.
Five (5) nanograms of total RNA was used as input for cDNA synthesis using the Clontech SMART-Seq v4 Ultra Low Input RNA kit (Takara Bio) according to the manufacturer’s instructions. Amplified cDNA was fragmented and appended with dual-indexed bar codes using the NexteraXT DNA Library Preparation kit (Illumina). Libraries were validated by capillary electrophoresis on an Agilent 4200 TapeStation, pooled at equimolar concentrations, and sequenced on an Illumina HiSeq3000 at 100SR, yielding 20-25 million reads per sample.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 3000 |
| |
|
| Data processing |
Illumina bcl2fastq v2.17.1.14 was used for demultiplexing.
Reads were mapped to the Rhesus macaque (MacaM v7) genomic reference with STAR (v2.5.2b) with default alignment parameters.
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: MacaM v7 (MacaM_Rhesus_Genome_v7.fasta,MacaM_Rhesus_Genome_Annotation_v7.8.2.gtf) https://www.unmc.edu/rhesusgenechip/index.htm#NewRhesusGenome
Supplementary_files_format_and_content: tab delimited text file containing normalized read counts for each sample (in columns) and each annotated transcript (in rows).
|
| |
|
| Submission date |
Apr 08, 2020 |
| Last update date |
Apr 09, 2020 |
| Contact name |
Gregory K Tharp |
| E-mail(s) |
gktharp@emory.edu
|
| Phone |
404-727-7797
|
| Organization name |
Yerkes National Primate Research Center
|
| Department |
Developmental and Cognitive Neuroscience
|
| Lab |
Genomics Core
|
| Street address |
954 Gatewood Dr
|
| City |
Atlanta |
| State/province |
GA |
| ZIP/Postal code |
30329-4208 |
| Country |
USA |
| |
|
| Platform ID |
GPL23804 |
| Series (1) |
| GSE148290 |
Delineation and modulation of the natural killer cell transcriptome in rhesus macaques during ZIKV and SIV infections |
|
| Relations |
| BioSample |
SAMN14558577 |
| SRA |
SRX8081943 |
