|
| Status |
Public on Jan 06, 2023 |
| Title |
BM-RNA-187_TK3_V1-deleted-DNA/DNA/ALVAC/ALVAC+gp120_Week 13 |
| Sample type |
SRA |
| |
|
| Source name |
PBMCs/CD14+ cells
|
| Organism |
Macaca mulatta |
| Characteristics |
animal id: TK3
vaccination: V1-deleted-DNA/DNA/ALVAC/ALVAC+gp120
time point: post ALVAC+gp120
week: Week 13
protocol: VB-026
study: Study 2
time of acquisition: 11
|
| Treatment protocol |
The isolation of the CD14+ cells was performed from freshly isolated PBMCs (30x10^6) collected at weeks 13 in 12 vaccinated animals. The isolation was performed by using non-human primate CD14 MicroBeads (#130-091-097, Miltenyi Biotec) and following manufacturer instructions. Briefly, 30x10^6 Ficoll Plaque isolated PBMCs were incubated with 60 µl microbeads and 240 µl buffer at 4ºC for 15 min. At the end of incubation, cells were washed with 3 ml buffer and resuspended in 500 µl of buffer. Positive selection was performed using the AutoMACSpro (Miltenyi Biotec) using the Possel program (positive selection). At the end of the separation, cells were resuspended in R10 (RPMI media, 10% FBS and 1X antibiotic/antimycotic; GIBCO) and counted for the ATAC-seq assay (50,000 cells). Leftover cells were resuspended in 500 µl RNAlater (Invitrogen, Waltham, MA) and stored at -80ºC.
|
| Growth protocol |
Twelve juvenile (average age 3.91 years, 0.155 SD), female macaques were enrolled in the study. All twelve macaques were immunized twice with V1-deleted DNA-SIV at weeks 0 and 4. At weeks 8 and 12 all macaques were boosted with intramuscular inoculations of 10^8 Plaque Forming Units (PFU) of recombinant ALVAC (vCP2432). At week 12, vaccinated macaques also received 400 μg of v1-delted SIVmac251–M766 protein formulated in alum Alhydrogel. The isolation of CD14+ cells was performed from freshly isolated PBMCs collected at weeks 13. PBMCs were isolated from freshly collected blood.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
RNA isolation was performed using RNeasy Micro Kit (Cat#74004, Qiagen) following manufacturer instructions. Briefly, CD14+ cells in RNAlater were thawed on ice, 500 µl PBS was added to each vial, and centrifuged at 3000 rpm for 10 min at 4ºC. Supernatants were discarded while cell pellets were processed following manufacturer protocol and resuspended in a final volume of 14 µl of water.
The library preparation was done using the NEBNext Ultra II Directional RNA Library Prep for Illumina kit (cat # E7760L, New England BioLabs) and following manufacturer instructions. The final product was quantitated by qPCR before cluster generation and sequencing.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Data processing |
average number of reads of 114 million/sample were acquired with a read length of 151 bp and with a paired-end read
Raw sequencing data were demultiplexed by Bcal2fastq v2.17. Adaptor sequence trimming was done by Cutadapt 1.18.
The trimmed RNA-Seq reads were aligned against rheMac10 using STAR 2.7.
Raw counts of mapped reads were obtained using the featureCounts program. Normalized data using default DESeq2 size factors were used for further analysis.
Supplementary_files_format_and_content: rheMac10
|
| |
|
| Submission date |
Nov 17, 2021 |
| Last update date |
Jan 06, 2023 |
| Contact name |
Massimiliano Bissa |
| E-mail(s) |
massimiliano.bissa@nih.gov
|
| Phone |
2407606578
|
| Organization name |
National Institutes of Health
|
| Department |
NCI/CCR
|
| Lab |
AMRVS
|
| Street address |
9000 Rockville Pike, bldg 41/ room c303
|
| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
| |
|
| Platform ID |
GPL27943 |
| Series (1) |
| GSE189032 |
RNA-seq profiling in CD14+ cells isolated from rhesus macaques vaccinated with V1-deleted DNA/ALVAC/gp120 vaccine |
|
| Relations |
| BioSample |
SAMN23242999 |
| SRA |
SRX13158406 |
