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Sample GSM8197730

Query DataSets for GSM8197730

Status

Public on Apr 16, 2024

Title

B6AIDKOWT321

Sample type

SRA

Source name

lymphoma

Organism

Mus musculus

Characteristics

tissue: lymphoma
strain: C57BL/6
genotype: AID-knockout B6
treatment: TBLV-WT

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

genomic DNA

Extraction protocol

For MiSeq sequencing, genomic DNA extracted from tumors induced by TBLV-WT and TBLV-SD at the higher dose (3 tumors each) in wild-type and Aicda-/- B6 mice was used for PCR with primers env7254(+) (5’-ATC GCC TTT AAG AAG GAC GCC TTC T-3’) and LTR9604(-) (5’-GGA AAC CAC TTG TCT CAC ATC-3’), resulting in ~2 kb amplicon of the envelope-LTR junction region. Reactions were performed with JumpStart RED Accutaq LA polymerase (Sigma-Aldrich) in the supplied buffer, 1 µg of tumor DNA, 50 pmol of each primer, and 0.5 mM deoxynucleoside triphosphates in 20 µl. PCR parameters were: 94oC for 1 min, then 10 cycles at 94oC for 10 sec, 53oC for 30 sec, and 68oC for 2 min followed by 25 cycles of 95oC for 15 sec, 50oC for 30 sec, and 68oC for 2 min with a final incubation at 68oC for 7 min. Sixteen independent reactions for each tumor sample were pooled, and DNA was sheared to a size of approximately 400 bp using a Covaris ultrasonicator at the UT Austin Genomic Sequencing and Analysis Facility (GSAF). For Nanopore sequencing, genomic DNA extracted from tumors from B6, B6 AID (Aicda)-knockout, or B6 mA3-AID double-knockout mice induced by TBLV-WT or TBLV-SD was used for PCR with primers env7254(+) (5’-ATC GCC TTT AAG AAG GAC GCC TTC T-3’) and LTR9604(-) (5’-GGA AAC CAC TTG TCT CAC ATC-3’) for the region spanning the envelope-LTR junction. PCRs were performed in 25 µl with JumpStart RED Accutaq LA polymerase (Sigma-Aldrich, cat# D8045) in the supplied buffer, 500 ng of tumor DNA, 25 to 50 pmol of each primer, and 0.5 mM deoxynucleoside triphosphates. PCR parameters were: 94oC for 1 min, 10 cycles at 94oC for 10 sec, 53oC for 30 sec, 68oC for 2 min followed by 25 cycles of 95oC for 15 sec, 50oC for 30 sec, and 68oC for 2 min with a final incubation at 68oC for 7 min. The fragment size for the env-LTR junction was ~2kb.
For MiSeq sequencing, fragmented DNA was end repaired and used for dA-tailing and ligation with Illumina adapters (unique to each tumor sample). Adapter-ligated DNA was amplified using these PCR parameters: 98oC for 30 sec, 6 cycles at 98oC for 10 sec, 65oC for 30 sec, and 72oC for 30 sec with a final incubation at 72oC for 5 min. Library preparations were submitted for sequencing on the MiSeq platform at the UT Austin Genome Sequencing and Analysis Facility (GSAF). Nanopore sequencing was employed for high-throughput sequence analysis of TBLV proviruses from the tumors induced by low-dose infection. The extracted PCR fragment used for Sanger sequencing was purified using a Zymoclean Gel DNA Recovery Kit (Zymo Research, cat# D4002). DNA (~1 µg) was prepared for Nanopore long amplicon sequencing by the University of Wisconsin Biotechnology Center.

Library strategy

OTHER

Library source

genomic

Library selection

other

Instrument model

MinION

Description

AID-knockout B6 mouse TBLV-WT derived tumor
snp_counts_nanopore.csv

Data processing

*library strategy: DNA amplicon
Aligned against provided reference using BWA mem
Duplicates marked using Picard MarkDuplicates
Marked duplicates filtered out using samtools
Mapped read counts quantified by position using bam-readcount (https://github.com/genome/bam-readcount)
Resulting positional counts analyzed using custom Python and R scripts
Alternative base frequencies were dichotomized using a cutoff of 3%.For each pair (reference base to alternate base), the dichotomized alternate calls were modeled as a function of sample group (TBLV-SD versus TBLV-WT in wild-type, AID-knockout, or mA3/AID double-knockout B6 mice).A mixed-effects logistic regression approach was used, incorporating a random effect that accounted for sample variation within the group.
Assembly: Custom TBLV (T-cell tropic MMTV) sequence
Supplementary files format and content: csv file containing readcount results

Submission date

Apr 11, 2024

Last update date

Apr 16, 2024

Contact name

Anna Battenhouse

Organization name

The University of Texas at Austin

Department

Center for Biomedical Research Support

Lab

Bioinformatics Consulting Group