title: LP/QE1
organism: Mus musculus
description: Protocols: All experiments were performed on 8-10 weeks old C57BL/6 mice (The Jackson Laboratory) and procedures conformed to ethical principles and guidelines revised and approved by the animal care committee of Gustave Roussy. Red cells were lysed from splenic suspensions for 5 minutes at 4°C. After washing (PBS 1X; 2% FBS), CD43- cells were separated using AutoMACS Pro Separator instrument (Miltenyi) with anti-CD43 antibody coated magnetic beads and comprised more than 95% B220+ cells (Supplementary Fig. S5c). CD43- cells were activated by in vitro stimulation with anti-IgM (7 µg/ml) and anti-CD40 (10 µg/ml) for 24h before transduction with the retroviral vectors, and transduced B cells were cultured in media containing anti-CD40 (10 µg/ml) for 36h after spin-infection. The number of viable cells was determined by counts of Trypan blue-negative cells and the percentage of GFP+ Sytox- cells was determined by flow cytometry. Washed, transduced cells were then cultured in 48-well plates at 106 cells/ml in fresh complete RPMI without any mitogen supplement. The start of the mitogen-free cultures was designated as “H0”. The number of GFP+ cells during culture was determined by counting Trypan blue-negative cells and measure the percentage of GFP+ cells, CD19+ and/or CD138+ GFP+ cells by flow cytometry. Lentiviral pV81 vectors with doxycycline inducible expression of WT or mutant SPI1 fused to GFP (pV81/GFP-SPI1) and GFP alone were used to transform cells. The pV81 backbone was described in this reference : Calvo J, BenYoucef A, Baijer J, Rouyez MC, Pflumio F. Assessment of human multipotent hematopoietic stem/progenitor cell potential using a single in vitro screening system. PLoS One. 2012;7:e50495. The sorted cells were subjected to DNA/RNA extraction using the AllPrep DNA/RNA Kit (Qiagen). RNA-seq was performed as described in Damm F, Mylonas E, Cosson A, Yoshida K, Della Valle V, Mouly E, et al. Acquired initiating mutations in early hematopoietic cells of CLL patients. Cancer Discov 2014;4(9):1088-101 doi 10.1158/2159-8290.
attributes.tag: ENA-FIRST-PUBLIC
attributes.value: 2019-05-03T04:03:17Z
attributes.tag: ENA-LAST-UPDATE
attributes.value: 2019-04-23T13:55:18Z
attributes.tag: External Id
attributes.value: SAMEA5574684
attributes.tag: INSDC center name
attributes.value: INSERM U1170, Gustave Roussy, Villejuif, France
attributes.tag: INSDC first public
attributes.value: 2019-05-03T04:03:17Z
attributes.tag: INSDC last update
attributes.value: 2019-04-23T13:55:18Z
attributes.tag: INSDC status
attributes.value: public
attributes.tag: Submitter Id
attributes.value: E-MTAB-7882:LP/QE1
attributes.tag: age
attributes.value: 8 to 10
attributes.tag: broker name
attributes.value: ArrayExpress
attributes.tag: cell type
attributes.value: B cell
attributes.tag: common name
attributes.value: house mouse
attributes.tag: genotype
attributes.value: MYD88-LP, pV81/GFP-SPI1-QE
attributes.tag: organism part
attributes.value: spleen
attributes.tag: sample name
attributes.value: E-MTAB-7882:LP/QE1
attributes.tag: scientific_name
attributes.value: Mus musculus
attributes.tag: strain
attributes.value: C57BL/6