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| Status |
Public on Mar 23, 2020 |
| Title |
Mock_4 [IonXpress_042] |
| Sample type |
SRA |
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| Source name |
CD19+ splenocytes isolated from humanized NSG mice 5 wks post mock-infection
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| Organism |
Homo sapiens |
| Characteristics |
human fetal liver donor: B
infection group: Mock (EBV–)
treatment group: Mock (FK506–)
cohort# - animal#: 4-6
cell type: B cells
cell subtype: CD19+ splenocytes
macroscopic tumor: negative
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| Treatment protocol |
13 to 19 weeks after engraftment, huNSG-A2 mice were injected with 105 RIU of EBV (B95-8) or 100µl PBS intraperitoneally. Animals were treated with 10mg/kg body weight of FK506 (Prograph injectable solution, Astellas) diluted 1:3 in PBS subcutaneously every second day starting at day 22 p.i. or left untreated.
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| Growth protocol |
Newborn HLA-A2 transgenic NOD/LtSz-scid IL2Rgnull (NSG-A2) mice mice (up to five days old) were irradiated with 1 Gy. Five to seven hours after irradiation, mice were injected intrahepatically with 1-4×105 CD34+ human hematopoietic progenitor cells (HPCs) derived from human fetal liver (HFL) tissue. Individual mouse cohorts were reconstituted with cells derived from three different HFL donors (A-C). Reconstitution of human immune system components in mice was analyzed in the week before the start of the experiments by flow cytometry.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Single cell suspensions of huNSG-A2-derived splenocytes were subjected to lysis of erythrocytes by ACK lysing buffer (Life Technologies) followed by magnetoc cell sorting of B cells using CD19 microbeads (Miltenyi Biotech). Total RNA of CD19+ cells was isolated using the RNeasy Mini Kit (QIAGEN) and traces of genomic DNA were removed by on-column DNase digestion (Qiagen). Purified RNA was stored at −20°C.
RNA quality was assessed further by an Agilent 2100 Bioanalyzer using the RNA 6000 Nano Kit. All RIN numbers were ≥ 9.5. RNA was quantified by NanoDrop 1000 spectrophotometry. Reverse transcription of 10 ng total RNA, amplification of targets by ultra-high multiplex PCR, and sequencing library construction was performed using the Ion AmpliSeq Transcriptome Human Gene Expression Kit (Thermo Fisher Scientific; #A26325) and Ion Xpress barcodes. Unamplified libraries were quantified by qPCR and equal amounts were combined for sequencing (6 µl of 100 pM total libraries). Templating of Ion Sphere Particles by emulsion PCR was conducted using the Ion 540 Kit (Thermo Fisher Scientific #A27753) and an OT2 instrument. Sequencing using an Ion 540 semiconductor chip was carried out on the Ion S5 System (Thermo Fisher Scientific).
The Ion AmpliSeq Transcriptome Human Gene Expression Kit uses primers targeting 18,574 mRNAs and 2,228 non-coding RNAs (ncRNAs) for a total of 20,802 transcripts. 10 ng total RNA per sample were processed, and IonXpress barcodes were used for library building.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Ion Torrent S5 |
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| Description |
IonXpress_042
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| Data processing |
Sequencing reads were mapped to Homo sapiens (human) genome assembly GRCh37, and initial analysis, quality control, and normalization of mapped reads as RPM (reads per million mapped reads) was performed by the ampliSeqRNA plug-in on the Ion Torrent server.
All subsequent statistical analysis of the transcriptome data was performed using the R environment for statistical computing (R version 3.4.1 (2017-06-30)) with Bioconductor (Huber W, Carey VJ, Gentleman R, Anders S, Carlson M, Carvalho BS, Bravo HC, Davis S, Gatto L, Girke T, et al. Orchestrating high-throughput genomic analysis with Bioconductor. Nat Methods. 2015;12(2):115-21) and dedicated packages.
To avoid technical artifacts the data were filtered such that genes for which the average over all samples was less than 1 were removed.
Identification and correction for spurious batch effects of unknown origin was performed via application of surrogate variable analysis (Leek JT, and Storey JD. Capturing heterogeneity in gene expression studies by surrogate variable analysis. PLoS Genet. 2007;3(9):1724-35.)
Differential expression was modeled using mixed-effect linear regression with empirical Bayes variance estimation, as implemented in the limma package (3.34.3) (Ritchie ME, Phipson B, Wu D, Hu Y, Law CW, Shi W, and Smyth GK. limma powers differential expression analyses for RNA-sequencing and microarray studies. Nucleic Acids Res. 2015;43(7):e47.)
Mapping of the transcripts to HGNC symbols and ENTREZID’s was performed using the org.Hs.eg.db package version 3.5.0.
Genome_build: hg19
Supplementary_files_format_and_content: Tab-delimited text files include gene symbol and gene expression (RPM) for each sample
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| Submission date |
Feb 13, 2019 |
| Last update date |
Mar 25, 2020 |
| Contact name |
Christian Münz |
| E-mail(s) |
muenzc@immunology.uzh.ch
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| Organization name |
University Of Zurich
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| Department |
Institute of Experimental Immunology
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| Lab |
Viral Immunobiology
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| Street address |
Winterthurerstrasse 190
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| City |
Zurich |
| ZIP/Postal code |
8057 |
| Country |
Switzerland |
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| Platform ID |
GPL23934 |
| Series (1) |
| GSE126515 |
AmpliSeq Transcriptome profiling of human B cells from EBV-infected and FK506-treated humanized mice. |
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| Relations |
| BioSample |
SAMN10925074 |
| SRA |
SRX5372078 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3602861_Mock_4_processed_RPM.txt.gz |
104.6 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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