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SRX24213414: GSM8196060: AidWT_RemSD_b46b_2; Mus musculus; RNA-Seq
2 ILLUMINA (Illumina NovaSeq 6000) runs: 3.1M spots, 315.3M bases, 82.3Mb downloads

External Id: GSM8196060_r1
Submitted by: Bioinformatics Consulting Group, Center for Biomedical Research Support, The University of Texas at Austin
Study: Apobec-Mediated Retroviral Hypermutation In Vivo is Dependent on Mouse Strain [RNA-seq]
show Abstracthide Abstract
Retroviruses cause lifelong infections resulting from their ability to thwart innate immunity. The Apobec family of cytidine deaminases are part of the innate immune response that recognizes and mutates foreign nucleic acids, including those from multiple viruses. Multiple retroviral antagonists of Apobecs have been identified, including mouse mammary tumor virus (MMTV)-encoded Rem protein. Previous experiments have shown that Rem-null MMTV or closely related TBLV proviruses from BALB/c tumors accumulate G-to-A and C-to-T mutations typical of Apobecs compared to wild-type proviruses expressing Rem. The difference in mutations between Rem-expressing and non-expressing MMTV strains largely disappeared in mice lacking the Apobec family member, activation-induced cytidine deaminase (AID). These results suggested that Rem is an AID antagonist. In this study, we attempted to study AID-mediated mutations of TBLV proviruses lacking Rem expression obtained from tumors in C57BL/6 (B6) wild-type and AID-knockout backgrounds. Surprisingly, no differences in G-to-A mutations were observed in TBLV proviruses regardless of Rem expression, yet such mutations were significantly reduced in proviruses obtained from mA3/AID-double knockout mice relative to those from wild-type B6 or AID-knockout mice. Many cellular mRNAs involving the innate immune response, but not Apobecs, were elevated in the absence relative to the presence of Rem expression on the B6 AID-knockout background. These results revealed that Apobec-mediated mutagenesis is dependent on mouse strain and suggested a second means of Rem-dependent immune evasion. Overall design: Each of two mouse mammary tumor virus (MMTV) strains (TBLV-WT = Rem-expressing or TBLV-SD = lacks Rem expression) was used to generate tumors in C57BL/6 (B6) mice or AID-knockout B6 mice. For RNAseq, we extracted four or five tumors induced by infection with TBLV-WT (Rem+) or TBLV-SD (Rem-) either in wild-type B6 mice or AID-knockout mice. Total RNA was extracted separately from each tumor for analysis and used for library preparation prior to sequencing.
Sample: AidWT_RemSD_b46b_2
SAMN40918624 • SRS20983618 • All experiments • All runs
Organism: Mus musculus
Library:
Name: GSM8196060
Instrument: Illumina NovaSeq 6000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC
Selection: cDNA
Layout: SINGLE
Construction protocol: Library preparation and sequencing for TagSeq, a form of 3' RNA sequencing were performed by the Genomic Sequencing and Analysis Facility (GSAF) at The University of Texas at Austin. Total RNA was isolated from each sample (0.375 mL) by addition of Trizol (1.125 mL; Thermo Fisher) and the sample (1.4 mL) was transferred to a Phasemaker tube (Thermo Fisher). Total RNA was extracted following the protocol supplied by the manufacturer and further cleaned up using a RNeasy MinElute Cleanup Kit (QIAGEN). RNA integrity number (RIN) was measured using an Agilent Bioanalyzer and 100 ng of RNA was used for the Tag-Seq protocol. The fragmentation/RT mix was prepared then added to each RNA sample and then heated to 95 degrees for 2.5 minutes on a Thermocyler then immediately put on ice for 2 minutes. After cooling and addition of the template switching oligo and SmartScribe RT the fragmented RNA reaction was incubated 42°C for 1hr, 65°C for 15 min. Next an AmPure bead clean-up was completed for the cDNA before it was amplified for 18 cycles, also incorporating the Illumina sequencing primer site, followed by another cleanup. The remaining portions of the Illumina adapater (the i5 and i7 indices) were then incorporated through an additional 4 cycles of PCR. Final libraries were quantified with PicoGreen then pooled equally for size selection using the Blue Pippin from 355-550 bp.
Runs: 2 runs, 3.1M spots, 315.3M bases, 82.3Mb
Run# of Spots# of BasesSizePublished
SRR286151361,557,216157.3M41.1Mb2024-04-15
SRR286151371,564,149158M41.2Mb2024-04-15

ID:
32525113

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