Gene expression changes in in vitro equivalents of DC progenitors from uninfected or LCMV Cl13-infected mice upon Gmeb1 knockdown and treatment with glucocorticoid

Gmeb1 and glucocorticoids suppress pDC development. To address which genes are involved in regulation of pDC development downstream of Gmeb1 and/or glucocorticoids, we investigated the gene expression profiles of pro-DCs (in vitro equivalents of DC progenitors) from bone marrow Flt3L culture from uninfected or LCMV Cl13-infected mice after knockdown of Gmeb1 or treatment with corticosterone. Overall design: 7 weeks old C57Bl/6J mice were intravenously infected with 2x10^6 plaque forming units of lymphocytic choriomeningitis virus strain Clone-13. Bone marrow cells from 4-5 pooled mice were harvested to generate one single replicate per group. Bone marrow cells were harvested in duplicates at 8 days post infection and cultured in the presence of 100ng/ml Flt3L. For Gmeb1 knockdown, BM cells were enriched by using an EasySep Mouse Streptavidin Rapidspheres isolation Kit and biotin-conjugated anti-CD11b. Franctions unbound to streptavidin beads were cultured in the presence of Flt3L, and at day 2 post culture, the culture was enriched by using an EasySep Mouse Streptavidin Isolation Kit, biotin-conjugated anti-Gr-1, anti-CD19, and anti-CD127. Fractions unbound to streptavidin beads were transduced with retroviral particles encoding Gmeb1-targeting or control shRNA at days 2 and 3 post culture. For corticosterone treatment, bone marrow cells were cultured in the presence of vehicle or 50nM corticosterone. At 3.5 days post culture, pro-DCs were defined as Lin-CD11b-/loCD11c- cells (Lin includes B cells (CD19, B220), T cells (Thy1.2, CD3, CD4, CD8, CD127), NK cells (NK1.1), red blood cells (Ter119), granulocytes (Gr-1), monocytes (CD11b), and dendritic cells (MHCII)) were purified by using fluorescence-activated cell sorting (FACS). Pro-DCs transduced with retroviruses encoding control or Gmeb1-targeting shRNA were subsequently analyzed on a HiSeq 4000 instrument using a single read 100bp protocol. Pro-DCs treated with vehicle or 50nM corticosterone was sequenced using NOVA-seq with a paired end 50bp protocol.