miRNomes of haematopoietic stem cells and dendritic cells identify miR-30b as a regulator of Notch1

Su, Xiaoping; Qian, Cheng; Zhang, Qian; Hou, Jin; Gu, Yan; Han, Yanmei; Chen, Yongjian; Jiang, Minghong; Cao, Xuetao

Identification of new miRNA candidates. We next identified new miRNA genes among the unannotated sequences in the processes of DC development and differentiation. A total of 53 new miRNA candidates were identified by 'MIREAP' algorithm Table S2) 27 . Five novel miRNAs (DCm009, DCm015, DCm024, DCm033 and DCm044) were also confirmed to exist in the in vivo counterpart of mature DCs by Illumina sequencing. All new miRNA candidates were confirmed by qRT-PCR analysis in order to independently validate their existence, and 40 of them were validated by qRT-PCR in myelogenous cell line RAW264.7 (RAW) cells (Fig. 4a and Supplementary Table S2 ). These new miRNA candidates were expressed at very low levels. To investigate whether these new miRNA candidates were silenced by epigenetic modifications, we detected the miRNA expression in RAW cells upon treatment with HDAC inhibitor Trichostatin A (TSA). A total of 24 new miRNA candidates were upregulated upon TSA treatment, and miR-1-1 was used as positive control (Fig. 4a and Supplementary Table S2) 28 . ChIP assays on the potential promoter of representative candidates (DCm009, DCm019, DCm020, DCm021 and DCm023), using two sets of qPCR primers spanning the promoter (Fig. 4b) , showed upregulation of histone H3 acetylation in TSA treatment group (Fig. 4c,d) , miR-1-1 was used as positive control.