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Status |
Public on Apr 15, 2024 |
Title |
AidWT_RemSD_b46b_2 |
Sample type |
SRA |
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|
Source name |
lymphoma
|
Organism |
Mus musculus |
Characteristics |
tissue: lymphoma strain: C57BL/6 genotype: Aicda+/+ treatment: infected with a mutant MMTV strain defective in Rem and its cleavage product Rem-CT
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Treatment protocol |
For each strain of mice, age-matched weanlings 4-to-6 weeks of age were injected intraperitoneally with either 1 x 106 (low dose) or 2 x 107 (high dose) stably transfected Jurkat T cells. Infectious clones were used for transfection of Jurkat cells and selected for three weeks in hygromycin and then screened for production of equivalent amounts of TBLV-WT or TBLV-SD (Rem-null) as judged by Western blotting with antibodies to MuLV capsid protein. Mice were monitored for the development of thymic and/or splenic tumors for a period of 9-12 months
|
Extracted molecule |
polyA RNA |
Extraction 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.
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Library strategy |
RNA-Seq |
Library source |
transcriptomic |
Library selection |
cDNA |
Instrument model |
Illumina NovaSeq 6000 |
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|
Description |
5' mRNA C57BL/6 mouse lymphoma cells induced by Rem/TBLV defective MMTV B6SD-46B-2
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Data processing |
TagSeq (5' mRNA-seq) preprocessing was performed in order to collapse duplicates based on defined TagSeq molecular indices (swMWWM, swGC, swTG, swMW) plus the adjacent 24 bases. Reads with no matching TagSeq tag were discarded. Adapter sequences (AGATCGGAAG) and PolyA tails (AAAAAAAA+) were removed. Resulting sequences shorter than 20 bases were discarded, as were sequences with more than 10% of the remaining bases having base qualities less than 10. Single-end pseudo alignment was performed using kallisto (ver. 0.45.0, options --single-overhang --bias --fragment-length 200) against the mouse transcriptome (GENCODE M26 transcript sequences). Downstream analysis of kallisto estimated counts was performed in R (ver. 4.0.3). The tximport package (v1.18.0) was used to roll up transcript-level counts into gene-level counts provided to the DESeq2 package (ver. 1.30.0). Before DESeq2 analysis, count data matrices were filtered to remove genes with fewer than 1 read across all included samples. The DGE models analyzed included Rem SD vs Rem WT in both AID WT and AID KO tumors Assembly: GENCODE M26 transcriptome (transcript sequences and main gene annotation set) Supplementary files format and content: Tab-delimited kallisto_counts.tsv text files provide kallisto per-transcript estimated counts and transcripts per million values for each sample. Full DESeq2 DGE analysis details can be found in the Supplementary zip file.
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|
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Submission date |
Apr 10, 2024 |
Last update date |
Apr 15, 2024 |
Contact name |
Anna Battenhouse |
Organization name |
The University of Texas at Austin
|
Department |
Center for Biomedical Research Support
|
Lab |
Bioinformatics Consulting Group
|
Street address |
100 East 24th St., Stop A5000
|
City |
Austin |
State/province |
TX |
ZIP/Postal code |
78712 |
Country |
USA |
|
|
Platform ID |
GPL24247 |
Series (1) |
GSE263655 |
Apobec-Mediated Retroviral Hypermutation In Vivo is Dependent on Mouse Strain [RNA-seq] |
|
Relations |
BioSample |
SAMN40918624 |
SRA |
SRX24213414 |