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| Status |
Public on Feb 24, 2022 |
| Title |
2_89-12R-pSCM |
| Sample type |
SRA |
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| Source name |
CD4+ Tcells
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| Organism |
Macaca mulatta |
| Characteristics |
subject id: 89-12R
Sex: Female
cell type: stem cell memory CD4+ T-cells (SCM)
treatment: Treated
dose: 10mg
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| Treatment protocol |
Twelve Indian rhesus macaques (RMs), including 5 males and 7 females, were infected i.v. with 103 TCID50 of SIVmac251. Starting at day 11 post-infection, all 12 animals were initiated on triple ART consisting of two reverse transcriptase inhibitors (PMPA/tenofovir and FTC/emtricitabine) and one integrase inhibitor (dolutegravir). After 14-15 weeks on ART and plasma viral load suppression <80 copies/ml for at least 4 weeks, 8 RMs additionally received the CBP/β-catenin inhibitor PRI-724 while the 4 remaining RMs were maintained on ART only and served as controls (Figure 1). Among the PRI-724-treated group, 5 RMs received 6 cycles (one week on/one week off) of PRI-724 at 10mg/kg/day administered subcutaneously (s.c.). Based on results from a concurrent dose ranging study (Fig. S1), an additional 3 RMs received 12 weeks of uninterrupted PRI-724 at 20mg/kg/day s.c., a dose that was found to be safe in healthy RMs.
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| Extracted molecule |
total RNA |
| Extraction protocol |
After isolation, cells were resuspended in PBS containing 2 mM EDTA and spun for the removal of contaminating platelets. Prior to sorting, peripheral CD4+ T-cells were enriched with the use of magnetic beads and column purification (Miltenyi Biotec). Enriched peripheral CD4+ T-cells were then stained with previously determined volumes of the following fluorescently conjugated mAbs: CD3-APC-Cy7 or -AF700 (clone SP34-2), CCR7-PE-Cy7 (clone 3D12), CD8-APC-Cy7 (clone SK1), CD45RA-APC (clone 5H9), CD95-PE-Cy5 (clone DX2), (CD62L-PE (clone SK11) from BD Bioscience; CD28-ECD (clone CD28.2) from Beckman Coulter, CD4-BrilliantViolet650 (clone OKT4), CD8-BV421 (clone RPA-T8) from Biolegend. Circulating populations for sorting were defined as follows: naive, CD45RA+CCR7+CD95-CD62L+; TSCM, CD45RA+CCR7+CD95+CD28+CD62L+; TCM, CD45RA-CD95+CCR7+CD62L+; and TEM, CD95+CCR7−. RNA was purified using QIAGEN Micro RNEasy columns, and RNA quality assessed using Agilent Bioanalyzer.
10 ng of total RNA was used as input for mRNA amplification using 5’ template-switch PCR with the Clontech SMART-Seq v4 Ultra Low Input RNA kit, according to manufacturer’s instructions. Amplified mRNA was fragmented and appended with dual indexed bar codes using Illumina NexteraXT DNA Library Prep kits. Libraries were validated by capillary electrophoresis on an Agilent 4200 TapeStation, pooled, and sequenced on an Illumina HiSeq 3000 using (151 bp SR) at an average read depth of 23 M.
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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 |
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)
Supplementary_files_format_and_content: tab delimited text file containing normalized read counts for each sample (in columns) and each annotated transcript (in rows).
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| Submission date |
Feb 27, 2019 |
| Last update date |
Feb 24, 2022 |
| Contact name |
Gregory K Tharp |
| E-mail(s) |
gktharp@emory.edu
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| Phone |
404-727-7797
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| Organization name |
Yerkes National Primate Research Center
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| Department |
Developmental and Cognitive Neuroscience
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| Lab |
Genomics Core
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| Street address |
954 Gatewood Dr
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| City |
Atlanta |
| State/province |
GA |
| ZIP/Postal code |
30329-4208 |
| Country |
USA |
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| Platform ID |
GPL23804 |
| Series (1) |
| GSE127330 |
Pharmacological modulation of the Wnt/β-catenin pathway inhibits the proliferation of long-lived latently-infected memory CD4+ T-cells in ART-suppressed SIV-infected macaques |
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| Relations |
| BioSample |
SAMN11030861 |
| SRA |
SRX5443705 |
| 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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