title: Illumina HiSeq 2000 paired end sequencing; Effect of MHC II expression in pro-B cells: F6 leukaemic cell line
design: Effect of MHC II expression in pro-B cells: F6 leukaemic cell line
library_construction_protocol: The F6 murine pro-B-cell leukaemia line was originally established from a spontaneous leukaemia in mice overexpressing IL-7 and has been previously described in Fisher, A. G., Burdet, C., Bunce, C., Merkenschlager, M. & Ceredig, R. Lymphoproliferative disorders in IL-7 transgenic mice: expansion of immature B cells which retain macrophage potential. Int. Immunol. 7, 415-423 (1995).  F6 cells were grown in culture in IMDM with 5% FCS and were transduced with pRV-based retroviral vectors expressing either GFP or MHC II (mouse H2-Aa and H2-Ab1 chains). Retroviral particles were produced by transfection of pRV plasmids encoding MHC II chains or GFP into the Platinum-E (Plat-E) retroviral packaging cell line, using the GeneJuice transfection reagent (Novagen). Plat-E culture supernatants containing viral stocks were harvested two days after the initial transfection and used for transduction of target cells. Cells were mixed with supernatants containing viral stocks in the presence of 4 μg/ml polybrene (Sigma-Aldrich) and spun at ~315×g for 45 min, before resuspended in IMDM for further culture. F6 cells were grown in culture in IMDM with 5% FCS F6 cells were transduced either with GFP or with MHC II RNA was then extracted using the RNeasy Mini QIAcube or Micro kits (Qiagen) Illumina, RNA with PolyA selection (at GeneWiz)
attributes.tag: Experimental Factor: phenotype
attributes.value: empty retroviral vector