title: KEM001
organism: Mus musculus
description: Protocols: 5T33 inoculated mice take approximately 3 weeks to develop MM while 5T2 inoculated mice take approximately 3-4 months. At that point, bone marrow was isolated by flushing the long bones. Briefly, bone marrow was flushed out, filtered using a nylon gause and subjected to red blood cell lysis. Samples were washed by PBS supplemented with 1% BSA and 0.01% azide. Next, cells were stained with anti-idiotype antibodies (in-house production) and anti-CD11b-FITC (ImTec Diagnostics, Antwerp, Belgium) for 30min at 4°C. Following washing with PBS/BSA/azide, the samples were incubated with the secondary antibody rat anti-muis IgG1-APC (BD Bioscience, Erembodegem, Belgium) for 30min at 4°C. After the last washing step, samples were dissolved in PBS/BSA azide supplemented with 2µl 7AAD (BD Bioscience) per million cells. Tumor cells were FACS sorted. To obtain >95% pure tumor populations, we gated out CD11b and 7AAD-positive cells followed by positive selection of anti-idiotype positive cells. C57BL/KaLwRij and C57BL/6 mice were purchased from Envigo (Horst, Nederland). Mice were housed according to the approved conditions by the Ethical Committee for Animal Experiments of the Vrije Universiteit Brussel (license no. LA1230281). 5T33 and 5T2 models were maintained as previously described (35). The 5TGM1 cell line was cultured at 37°C and 5% CO2 in RPMI-1640 medium (Lonza, Basel, Switserland) supplemented with 100U/ml penicillin/streptomycin, 1mM Na-pyruvate, 2mM glutamine (Gibco, Eggenstein, Germany) and 10% fetal calf serum (FCS, Hyclone, UT, USA). DNA from 5TGM1 cells, sorted 5T33 and 5T2, and tails was isolated using the Wizard Genomic DNA purification kit (Promega, Madison, WI, USA). DNA was dissolved in 80µl PBS + 4µl RNAse A and incubated for 10min at 37°C. After adding proteinase K and incubation for 10min at 56°C, DNA was purified using the Qiamp DNA mini kit according to manufactures instructions. (Qiagen) DNA was eluted in 100µl 10mM Tris and stored at -80°C for further processing. Whole-genome DNA libraries of 2 murine models, 1 cell line and 4 tails were prepared with Illumina's TruSeq DNA sample preparation kit V2 according to the manufacturer's instructions.
attributes.tag: ENA first public
attributes.value: 2018-10-09
attributes.tag: ENA last update
attributes.value: 2016-12-01
attributes.tag: External Id
attributes.value: SAMEA18629668
attributes.tag: INSDC center alias
attributes.value: VIB, Vesalius Research Center, Belgium VIB, Center for Cancer Biology KUL, Department of Oncology; Belgium
attributes.tag: INSDC center name
attributes.value: VIB, Vesalius Research Center, Belgium VIB, Center for Cancer Biology KUL, Department of Oncology; Belgium
attributes.tag: INSDC first public
attributes.value: 2018-10-09T17:03:35Z
attributes.tag: INSDC last update
attributes.value: 2016-12-01T21:14:35Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-5302:KEM001
attributes.tag: age
attributes.value: not available
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell line
attributes.value: 5TGM1
attributes.tag: common name
attributes.value: house mouse
attributes.tag: disease
attributes.value: multiple myeloma
attributes.tag: organism part
attributes.value: bone marrow (tumor)
attributes.tag: sample name
attributes.value: E-MTAB-5302:KEM001
attributes.tag: sex
attributes.value: female
attributes.tag: strain
attributes.value: C57BL/KaLwRij