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| Status |
Public on Jan 25, 2022 |
| Title |
LG11 30 minutes post-NKTT320 |
| Sample type |
SRA |
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| Source name |
PBMC
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| Organism |
Macaca fascicularis |
| Characteristics |
treatment: NKTT320 treated
time pointment: 30 minutes post-NKTT320
tissue: Blood
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| Growth protocol |
collection protocol: Whole blood was collected by venipuncture in EDTA vacutainers. Peripheral blood mononuclear cells (PBMCs) were isolated from MCM by density gradient centrifugation. In brief, peripheral blood was diluted 1:1 in PBS and layered over 90% ficoll at a 1:2 ratio. The gradient was then spun at 2,200 rpm for 45 minutes with the brake off. Cells were then isolated, washed and counted according to standard methods. Aliquots of 1M PBMC were pelleted, snap frozen and stored at -80C until RNA extraction.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from snap frozen, unfractionated PBMCs using the Qiagen RNeasy Plus Mini Kit (Qiagen 74134). Briefly, cells were first thawed and pelleted, then lysed following the kit protocol. gDNA was removed using a gDNA Eliminator spin column, and remaining flow through was added to a RNeasy spin column to bind RNA. The spin column/RNA was washed, RNA was eluted and stored at -80oC. Later, samples were concentrated using Zymo Clean & Concentrate-5 Kit (R1013). Briefly, RNA was thawed, bound, and added to the Zymo-Spin IC Column. Then RNA bound to the column was washed, eluted, aliquoted and stored at -80oC.
Samples were then submitted for RNA-Seq analysis using a high output, and single read 75 cycle run. Prior to Illumina total RNA library construction, DNase treated RNA was quantitated using the Qubit RNA BR assay kit (ThermoFisher Scientific, #Q10210). RNA quality (RIN) was determined on an Agilent 2100 Bioanalyzer using an Agilent RNA 6000 Nano kit (Agilent, #5067-1511). Cytoplasmic and mitochondrial rRNA was removed from each sample following the Ribo-Zero Gold rRNA Removal Kit Reference Guide from Illumina. After RNA clean up, samples were resuspended in 11ul Elution buffer followed by prime and fragmentation. Illumina compatible total RNA cDNA libraries were generated following the TruSeq Stranded Total RNA Sample Preparation Guide (Illumina #20020596).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
NextSeq 550 |
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| Data processing |
RNA-seq for Eukaryotes Analysis v3 was used for sequencing analysis and built by the Banana Slug Genomics Center at the University of California Santa Cruz. First, an Illumina sequencer at the Tulane School of Medicine Genomics Core was used to construct raw sequencing reads which were checked for quality and contaminants using FastQC. Next, Trimmomatic was used to trim adapter sequences and primers from the sequencing reads (Bolger et al., 2014). Removal of polyA tail, polyN, and read segments with a quality score below 28 was accomplished by using PRINSEQ (Schmieder and Edwards, 2011). Following trimming, any reads of length less than 20bp were removed. Quality check was repeated, and high-quality reads were then mapped to the GRCh37/hg19 reference genome using STAR (Kim et al., 2013, Dobin et al., 2013) with NCBI RefSeq (O'Leary et al., 2016) annotated genes transcriptome index data. Raw read counts were normalized across all samples and then used for differential expression analysis using DESeq (Anders and Huber, 2010) and edgeR (Robinson et al., 2010) separately. Genes related to the immune system and with p-values of <0.05 from the edgeR pipeline were identified and categorized by major cell type, which were then plotted on individual heatmaps. Heatmaps were constructed using log2 fold change data and using the ‘pheatmap’ package in R (Kolde, 2019).
GSEA was performed with the R package ‘ClusterProfiler’ at default parameters. Enrichment scores were calculated against the following 4 pathway databases containing a priori-defined gene sets: Gene Ontology (GO) database, Hallmark (H) and Curated (C2) gene sets of the Molecular Signature database (MsigDB), and WikiPathways. Gene sets significantly enriched in the datasets (p < 0.05) were subsequently curated for those relevant to NKT cell biological function. Enrichment plot was generated with the R software package ‘ggplot2’ and heatmaps with the ‘pheatmap’ package.
Genome_build: GRCh37/hg19 reference genome
Supplementary_files_format_and_content: tab delimited text files correspond to transcript read counts calculated by Htseq-count and fpkm-count
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| Submission date |
Jan 23, 2022 |
| Last update date |
Jan 26, 2022 |
| Contact name |
Nell G Bond |
| E-mail(s) |
[email protected], [email protected]
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| Phone |
6073512251
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| Organization name |
Tulane University School of Medicine
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| Street address |
Microbiology & Immunology, 1430 Tulane Avenue
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| City |
New Orleans |
| State/province |
LA |
| ZIP/Postal code |
70112 |
| Country |
USA |
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| Platform ID |
GPL27448 |
| Series (1) |
| GSE194235 |
Immunomodulatory potential of in vivo Natural Killer T (NKT) activation by NKTT320 in Mauritian-origin cynomolgus macaques |
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| Relations |
| BioSample |
SAMN25209044 |
| SRA |
SRX13876451 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5831347_LG11-30min-abundance.fpkm.txt.gz |
213.7 Kb |
(ftp)(http) |
TXT |
| GSM5831347_LG11-30min-abundance.htseq.txt.gz |
195.0 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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