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| Status |
Public on Oct 07, 2020 |
| Title |
R294: CFSE low CD4 high |
| Sample type |
SRA |
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| Source name |
Peripheral blood CD4+ T cells
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| Organism |
Erythrocebus patas |
| Characteristics |
treatment: SEB stimulation
tissue: Blood
cell type: Peripheral blood CD4+ T cells
dividing cells: CFSE low
cd69/cd4 expression: CD4 high
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| Treatment protocol |
For in vitro stimulation, peripheral blood mononuclear cell suspensions were labeled with CellTrace CFSE (ThermoFisher. Waltham, MA) and sorted for CD4+ T cells on a BD FacAria II. Cells were subsequently cultured in RPMI supplemented with 10% fetal bovine serum, 100 IU/ml Penicillin, 100 ug/ml Streptomycin, and 2.5mM glutamine (all from Gibco. Waltham, MA) in the presence of 1ug/ml Staphylococcal entrotoxin B (SEB) and autologous HLA-DR+CD11b+ antigen presenting cells. After 5 days of culture, responding cells were re-sorted on the basis of CD4 expression, CFSE dye dilution, and CD69 expression, lysed in 350ul RLT buffer supplemented with 1% b-2-mercaptoethanol (Qiagen. Germantown, MD) and preserved at -80C until further processing.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated following the Qiagen RNeasy Micro Kit protocol. Samples were purified through Qiagen columns with on-column DNase treatment for 15 minutes
. Libraries were made from purified total RNA using the Illumina TruSeq Stranded Total RNA Sample Preparation Kit protocol. rRNA was removed from samples by denaturing and then purified on rRNA removal beads. RNA was then fragmented and primed with random hexamers for first-strand synthesis in a single step on a thermocycler for 8 minutes at 94°C. First-strand synthesis immediately followed using Superscript II polymerase (Invitrogen) on a thermocycler with the following parameters: 25°C for 10mins, 42°C for 15 mins and 70°C 15 mins. In order to determine which strand the transcript was transcribed from, dUTP's were used instead of dTTP's during the second strand synthesis. Single adenines were added to the blunted ds cDNA. Illumina adapters containing unique dual-indices were ligated to each library. This allows the samples to be multiplexed on the sequencer. Libraries containing Illumina adapters were enriched using 15-18 cycles of PCR. Libraries were sequenced on an Illumina HiSeq 2500 (Illumina. San Diego, CA).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Files containing sequence reads and corresponding quality scores from all nonhuman primate samples were aligned to the Mmul_8.0.1 rhesus macaque genome assembly and annotation with STAR (ver2.7.3a) at default parameters (Dobin, Bioinformatics ’10). Bam files were sorted by genomic coordinate with SamTools (Li, Bioinformatics ’09) and subsequently counted with HTseq (Anders, Bioinformatics ’15). Parwise comparisons of gene counts were performed with DEseq2 (Love, Genome Biol ’14) and all genes with a logCPM < 30 were omitted from any downstream analysis. A p value of <0.01 when corrected for multiple comparison testing (Benjamini-Hochsberg) was used to define genes as significantly differentially expressed.
Genome_build: Mmul_8.0.1
Supplementary_files_format_and_content: tab delimited text files correspond to transcript read counts calculated by Htseq-count
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| Submission date |
Jun 04, 2020 |
| Last update date |
Jun 07, 2022 |
| Contact name |
Verena Link |
| E-mail(s) |
verena.link@nih.gov
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| Organization name |
NIH
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| Department |
NIAID
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| Street address |
4 Memorial Drive
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| City |
Bethesda |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL28632 |
| Series (2) |
| GSE151814 |
SIVagm coevolutions lead to altered epigenetic control of CD4 expression [RNA-seq] |
| GSE151815 |
SIVagm coevolutions lead to altered epigenetic control of CD4 expression |
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| Relations |
| BioSample |
SAMN15102156 |
| SRA |
SRX8472226 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4591273_JB01_count.txt.gz |
121.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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