title: wt_13148
organism: Mus musculus
description: Protocols: Primary FLC recipients were euthanized by cervical dislocation when reaching the humane endpoint specified by the experimental protocol. Lymphoma cells were isolated from lymph nodes and spleens and used for establishment of cell lines or further propagation by serial transplantation. Eµ-Myc (B6.Cg-Tg(IghMyc)22Bri/J) mice (44) were crossed with p53ERTAM/+ knock-in (Trp53tm1Gev, mixed C57BL/6 × 129/Sv background) animals (12) to generate Eµ-Myc;p53ERTAM/+ males that were further crossed with p53ERTAM/+ females to obtain Eµ-Myc;p53ERTAM/TAM embryos. To maintain an active p53 status during development of the hematopoietic lineage, pregnant mice were injected intraperitoneally (i.p.) with 50 µg/kg tamoxifen (TAM, Sigma-Aldrich) dissolved in corn oil (Sigma-Aldrich) starting from day E7.5. At E13.5, embryos where genotyped by PCR and fetal livers cells (FLCs) were isolated. Following lysis of red blood cells with ACK buffer (Thermo Fischer Scientific), FLCs were cultured in B cell medium (40% IMDM, 40% DMEM, 20% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin, 0.05 mM 2-Mercaptoethanol) supplemented with 0.2 ng/ml IL-3, 2 ng/ml IL-6, 20 ng/ml SCF (all cytokines from Immunotools) and 4% WEHI-3B supernatant. For establishment of p53-ON lymphomas all media were continuously supplemented with 100 nM 4-hydroxytamoxifen (4-OHT, Sigma-Aldrich). FLCs were transduced with luciferase-expressing retrovirus (pMSCV-Firefly-T2A-Gaussia) to enable longitudinal monitoring of disease progression. FLCs were plated in 6-well plates precoated with retronectin (40 µg/ml, Takara) and transduced with fresh virus supernatant (1 ml/well supplemented with 8 µg/ml polybrene) using spinoculation (25 min, 600xg, 37ºC). Cells were infected twice daily with 6 h interval for 2 days (in total 4 infections). Infected FLCs were transplanted into B6-albino mice (B6(Cg)-Tyrc-2J/J, Charles River) lethally irradiated with 7 Gy X-rays 6 hours before transplantation. Recipients were kept on neomycin-supplemented water (1.6 g/L, pH 3) starting 2 days before transplantation until 3 weeks after transplantation. Total RNA was extracted from lymphoma tissues and cultured cells was isolated using RNeasy Mini kit (Qiagen) according to manufacturer's instructions. For quality control of RNA extraction yield and Library synthesis products, RNA ScreenTape kit (Agilent technologies), D1000 ScreenTape kit (Agilent technologies), Qubit® RNA HS Assay kit (Invitrogen), Qubit® DNA HS Assay kit (Invitrogen) were used for each specific step purpose. RNA quality was assessed using the Experion RNA StdSens Analysis Kit (BioRad). RNAseq libraries were prepared from total RNA using the TruSeq Stranded total RNA LT kit (Illumina) according to the manufacturer's instructions. Quality of sequencing libraries was controlled on a Bioanalyzer 2100 using the Agilent High Sensitivity DNA Kit (Agilent). Pooled sequencing libraries were quantified with digital PCR (QuantStudio 3D, Thermo Fisher).
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-10-18T04:04:42Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-06-05T11:47:43Z
attributes.tag: External Id
attributes.value: SAMEA5693645
attributes.tag: INSDC center name
attributes.value: Institute of Molecular Oncology, Center for Tumor and Immunobiology, Philipps-University Marburg
attributes.tag: INSDC first public
attributes.value: 2019-10-18T04:04:42Z
attributes.tag: INSDC last update
attributes.value: 2019-06-05T11:47:43Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-8015:wt_13148
attributes.tag: age
attributes.value: 6 to 10
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: common name
attributes.value: house mouse
attributes.tag: developmental stage
attributes.value: adult
attributes.tag: disease
attributes.value: lymphoma
attributes.tag: sample name
attributes.value: E-MTAB-8015:wt_13148
attributes.tag: sampling site
attributes.value: neoplasm
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: sex
attributes.value: male
attributes.tag: strain
attributes.value: B6(Cg)-Tyrc-2J/J
attributes.tag: study group
attributes.value: p53 +/+
attributes.tag: transplanted cells genotype
attributes.value: E-Myc;53<tm1.1Thst> Trp53 +/+