Next generation sequencing of human monocyte derived dendritic cells stimulated with MGL-stimulating glycodendrimers in the absence or presence of LPS. Next generation sequencing of human monocyte derived dendritic cells stimulated with MGL-stimulating glycodendrimers in the absence or presence of LPS

To investigate the effect of MGL ligation in monocyte-derived dendritic cell biology, we generated control dendrimers and two different glycodendrimers exposing the MGL ligands αGalNAc or GalNAcβ1-4Gal. αGalNAc and GalNAcβ1-4Gal glycodendrimers were validated in the corresponding manuscript. Monocyte-derirved dendritic cells were generated by culturing monocytes for 4 days with IL-4 and GM-CSF. To study the effect of MGL-ligation at the transcriptional level, next generation sequencing was performed on monocyte-derived dendritic cells from three independent donors, stimulated for 4 h with control, αGalNAc or GalNAcβ1-4Gal glycodendrimers in the absence or presence of LPS. Overall design: 18 samples were included in next generation sequencing, including triplicates for control, αGalNAc and GalNAcβ1-4Gal glycodendrimers, all-in the absence or presence of LPS. The control dendrimers and control dendrimers in the presence of LPS serve as reference samples

为探究MGL凝集（MGL ligation）对单核细胞衍生树突状细胞（monocyte-derived dendritic cell）生物学特性的影响，我们制备了对照树枝状大分子（dendrimer），以及两种分别展示MGL配体αGalNAc或GalNAcβ1-4Gal的糖基化树枝状大分子（glycodendrimer）。上述αGalNAc与GalNAcβ1-4Gal糖基化树枝状大分子已在对应研究稿件中完成验证。

单核细胞衍生树突状细胞的制备方式为：将单核细胞与白细胞介素4（Interleukin-4, IL-4）及粒细胞-巨噬细胞集落刺激因子（Granulocyte-Macrophage Colony-Stimulating Factor, GM-CSF）共培养4天。

为从转录水平研究MGL凝集的生物学效应，我们对3名独立供体来源的单核细胞衍生树突状细胞进行了二代测序（next generation sequencing）；上述细胞分别经对照树枝状大分子、αGalNAc或GalNAcβ1-4Gal糖基化树枝状大分子刺激4小时，刺激体系中分别设置脂多糖（Lipopolysaccharide, LPS）存在与缺失两种条件。

实验整体设计如下：本次二代测序共纳入18份样本，涵盖对照、αGalNAc及GalNAcβ1-4Gal糖基化树枝状大分子处理组的生物学重复各3份，且每组均包含LPS存在与缺失两个亚组。其中，仅用对照树枝状大分子处理的样本，以及经LPS存在下的对照树枝状大分子处理的样本作为参考样本。