Illumina HiSeq 4000 sequencing; A new type of pathogenic transcription factor mutation targeting interferon regulatory factor 4 (IRF4) in human lymphoma
A new type of pathogenic transcription factor mutation targeting interferon regulatory factor 4 (IRF4) in human lymphoma
Splenic B cells were isolated from C57BL/6 mice by CD43 depletion with magnetic anti-mouse CD43 microbeads (Miltenyi Biotech, Cat#130-049-801) according to the manufacturer's instructions. To induce B cell activation and terminal differentiation, purified B cells (density: 1x106 cells/mL; 4x106 cells per well) were cultured in the presence of recombinant mouse IL-4 (25ng/mL; R&D; Cat#404-ML) and LPS (20µg/mL; Sigma; Cat#L2880) over night. 24h after isolation, B cells were collected (300xg, 5min, 4°C) and resuspended in B cell medium supplemented with 8µg/mL polybrene (EMD Millipore, #TR-1003) at a density of 2x106 cells/mL. To introduce IRF4 wildtype and mutant, 4x106 B cells per well were plated in 2mL on 6-well plates that had been coated with RetroNectin (Takara, #T100B; 25µg/mL; 4°C, overnight), blocked with 2% BSA (in PBS, 1h, RT) and pre-loaded with the respective retroviral particles (1h, 37°C). Retroviral transduction was performed by the addition of 2mL of the respective retroviral supernatant and subsequent centrifugation (800xg, 90min, 32°C). 24h after transduction, B cells were collected (300xg, 5min, 4°C), resupended in new B cell medium and cultured (density: 1x106 cells/mL; 4x106 cells per well) for another 72h (FACS for RNA-seq, flow cytometric analysis of plasma cell differentiation) in the presence of recombinant mouse IL-4 (25ng/mL; R&D; Cat#404-ML) and LPS (20µg/mL; Sigma; Cat#L2880). Nucleic acids were extracted as described in the accompanying manuscript Nucleic acids were extracted with RNeasy Micro Kit (Qiagen) per provider's protocol