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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Apr 24, 2023 |
| Title |
CD8_Prf1_Ruxo_2 |
| Sample type |
SRA |
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| Source name |
Blood
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6 Prftm1Sdz/J
tissue: Blood
cell type: CD8+ T cells
genotype: Prf1-/-
treatment: LCMV + Ruxolitinib
age: Day 9
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| Treatment protocol |
Prf1-/- mice were infected intraperitoneally with 2x105 plaque-forming units (PFU) of LCMV Armstrong (kindly provided by John Wherry, University of Pennsylvania, Philadelphia, PA). Mice were treated orally twice a day with 90 mg/kg ruxolitinib (kindly provided by Ross Levine, Memorial Sloan Kettering Cancer Center, New York, NY or from Incyte Corporation) starting day 4 post-infection. Ruxolitinib was dissolved in citrate buffer (0.1M, pH 3.5) with captisol (20% w/v). Anti-IFNg (aIFNg) neutralizing antibody (clone XMG1.2) (BioXCell) was administered peritoneally at 0.5 mg per mouse on days 4 and 7 post-infection (p.i.) or combined with ruxolitinib. Mice were euthanized and multiple HLH parameters were evaluated on day 9 p.i.
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from sorted cell populations using the RNeasy Micro Kit (Qiagen, Germantown, Maryland). The quality and quantity of the initial RNA was determined by using the Agilent 2100 Bioanalyzer with the Eukaryote Total RNA Pico kit.
For each good quality sample, 2.5-25 nanograms of total RNA was used in the Tecan Ovation RNA sequencing system v2 protocol as written to create SPIA (single primer isothermal amplification)-cDNA. Once the cDNA was purified, five hundred nanograms of each sample was sheared using the Covaris LE220 focused ultra sonicator. The shearing plate used was the 96 microtube-50 AFA fiber plate and the target base pair size was 300. The settings for the shearing were the following: Peak incident Power (W) of 450, duty factor of 15%, cycles per burst 1000 with a 100 second treatment time. Once the cDNA was sheared, libraries were created using the KAPA Hyper-Prep kit from Roche.
Based on the starting cDNA input amount, the Kapa Hyper protocol recommended four pcr cycles for the amplification step while the rest of the protocol was followed as described. The indexes used were the UDI DNA indexes from Illumina. Eukaryote Total RNA Pico kit (catalog: 5067-1513) Tecan (formally NuGen) Ovation RNA sequencing system v2 (catalog: 7102-32) Covaris 96 tube plate (catalog:520168) KAPA Hyper-Prep (catalog: KK8504) UDI DNA Indexes from Illumina (catalog: 20022370)
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
CD8_Prf1_Ruxo_2
2355250_9_CD8
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| Data processing |
Total stranded RNA sequencing data were processed by the internal AutoMapper pipeline. The raw reads were firs trimmed (Trim-Galore version 0.60), mapped to mouse genome assembly (mm10) (STAR v2.7). The gene level expression values were quantified (RSEM v1.31) based on GENCODE annotation (M22 [mm10]).
Assembly: mm10
Supplementary files format and content: counts matrix, RSEM counts
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| Submission date |
Nov 21, 2022 |
| Last update date |
Feb 28, 2024 |
| Contact name |
Kim Nichols |
| E-mail(s) |
kim.nichols@stjude.org
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| Organization name |
St. Jude Children's Research Hospital
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| Department |
Oncology
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| Lab |
Nichols Lab
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| Street address |
262 Danny Thomas Pl
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| City |
Memphis |
| State/province |
TN |
| ZIP/Postal code |
38105 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE218500 |
Cellular and transcriptional impacts of ruxolitinib versus IFN-gamma neutralization in mouse models of HLH |
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| Relations |
| BioSample |
SAMN31823131 |
| SRA |
SRX18337354 |
| 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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