Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Expression profiling of BMP9-stimulated mesenchymal stem cells

ABSTRACT: To identify potential target genes regulated by BMP9 in MSCs, we used microarray to profile expression patterns of BMP9- vs. GFP-stimulated MSCs. Subconfluent mouse C3H10T1/2 cells were transduced with adenoviral vector expressing BMP9 or GFP for 30h. Total RNA was isolated for microarray analysis using Affymetrix mouse MOE430A GeneChips. Experiments were done in triplicate.

ORGANISM(S): Mus musculus

SUBMITTER: Tong-Chuan He

PROVIDER: E-GEOD-48882 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Growth hormone synergizes with BMP9 in osteogenic differentiation by activating the JAK/STAT/IGF1 pathway in murine multilineage cells.

Huang Enyi E Zhu Gaohui G Jiang Wei W Yang Ke K Gao Yanhong Y Luo Qing Q Gao Jian-Li JL Kim Stephanie H SH Liu Xing X Li Mi M Shi Qiong Q Hu Ning N Wang Linyuan L Liu Hong H Cui Jing J Zhang Wenwen W Li Ruidong R Chen Xiang X Kong Yu-Han YH Zhang Jiye J Wang Jinhua J Shen Jikun J Bi Yang Y Statz Joseph J He Bai-Cheng BC Luo Jinyong J Wang Huicong H Xiong Feng F Luu Hue H HH Haydon Rex C RC Yang Li L He Tong-Chuan TC

Journal of bone and mineral research : the official journal of the American Society for Bone and Mineral Research 20120701 7

Growth hormone (GH) is usually released by somatotrophs in the anterior pituitary in response to the GH-releasing hormone and plays an important role in skeleton development and postnatal growth. However, it is unclear if extrapituitary GH exerts any effect on murine multilineage cells (MMCs). MMCs are multipotent progenitors that give rise to several lineages, including bone, cartilage, and fat. We have identified bone morphogenic protein 9 (BMP9) as one of the most osteogenic BMPs in MMCs by r ...[more]

PMID: 22467218

Publication: 1/2

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Project description:We generated C57BL/6 mice lacking Bmp10 and/or Bmp9 utilizing the Cre-loxP system. Briefly, Bmp9 constitutive deletion resulted from the replacement of exon 2 by a neomycin resistance cassette. Because Bmp10 deletion leads to early embryonic lethality, we used the tamoxifen-inducible Cre system to generate Bmp10-cKO mice (Rosa26-CreERT2;Bmp10lox/lox) by crossing Rosa26-CreERT2 mice with Bmp10lox/lox mice that possess loxP sites flanking exon 2. To generate double-KO (DKO) mice, we crossed these Rosa26-CreERT2;Bmp10lox/lox mice with Bmp9-KO mice. At the age of 8 weeks, mice were treated with tamoxifen (Sigma) by intraperitoneal injection once a day for 5 days at a dosage of 50 mg/kg. At the age of 5 months, Wild Type and DKO mouse lung tissue was flash frozen in liquid nitrogen and stored at -80°C. RNA extraction, RNA sample quality assessment, RNA library preparation, sequencing and raw data analysis were conducted at GENEWIZ, Inc. (South Plainfield, NJ, USA). Total RNA was extracted from frozen tissue using the Qiagen RNeasy Plus Mini kit. RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies, Carlsbad, CA, USA) and RNA integrity was checked with Agilent TapeStation (Agilent Technologies, Palo Alto, CA, USA). rRNA depletion was performed using Ribozero rRNA Removal Kit (Illumina, San Diego, CA, USA). RNA sequencing library preparation used NEBNext Ultra RNA Library Prep Kit for Illumina by following the manufacturer’s recommendations (NEB, Ipswich, MA, USA). Briefly, enriched RNAs were fragmented for 15 minutes at 94 °C. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3’ends, and universal adapter was ligated to cDNA fragments, followed by index addition and library enrichment with limited cycle PCR. Sequencing libraries were validated using the Agilent Tapestation 4200 (Agilent Technologies, Palo Alto, CA, USA), and quantified by using Qubit 2.0 Fluorometer (Invitrogen, Carlsbad, CA) as well as by quantitative PCR (Applied Biosystems, Carlsbad, CA, USA). The sequencing libraries were clustered on one lane of a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq 4000 instrument (or equivalent) according to manufacturer’s instructions. The samples were sequenced using a 2x150 Paired End (PE) configuration. Image analysis and base calling were conducted by the HiSeq Control Software (HCS). Raw sequence data (.bcl files) generated from Illumina HiSeq was converted into fastq files and de-multiplexed using Illumina's bcl2fastq 2.17 software. One mis-match was allowed for index sequence identification. After investigating the quality of the raw data, sequence reads were trimmed to remove possible adapter sequences and nucleotides with poor quality using Trimmomatic v.0.36. The trimmed reads were mapped to the the Mus musculus GRCm38 reference genome available on ENSEMBL using the STAR aligner v.2.5.2b. Gene counts were calculated from uniquely mapped reads using feature Counts from the Subread package v.1.5.2. Only unique reads that fell within exon regions were counted. The gene hit counts table was then used for downstream differential expression analysis. A differential gene expression analysis between WT and DKO groups of samples was performed using the R-package DESeq2 (Wald test).

Dataset Information

Expression profiling of BMP9-stimulated mesenchymal stem cells

Publications

Growth hormone synergizes with BMP9 in osteogenic differentiation by activating the JAK/STAT/IGF1 pathway in murine multilineage cells.

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