Unknown,Transcriptomics,Genomics,Proteomics

Dataset Information

Mus musculus hematopoietic stem cell

ABSTRACT: A transcriptome study in mouse hematopoietic stem cells was performed using a sensitive SAGE method, in an attempt to detect medium and low abundant transcripts expressed in these cells. Among a total of 31,380 unique transcript, 17,326 (55%) known genes were detected, 14,054 (45%) low-copy transcripts that have no matches to currently known genes. 3,899 (23%) were alternatively spliced transcripts of the known genes and 3,754 (22%) represent anti-sense transcripts from known genes. Mouse hematopoietic stem cells were purified from bone marrow cells using negative and positive selection with a Magnetic-Activated Cell Sorter (MACS). total RNA and mRNA were purified from the purified cells using Trizol reagent and magnetic oligo dT beads. Double strand cDNAs were synthesized using a cDNA synthesis kit and anchored oligo dT primers. After NlaIII digestion, 3’ cDNAs were isolated and amplified through 16-cycle PCR. SAGE tags were released from the 3’ cDNA after linker ligation. Ditags were formed, concatemerized and cloned into a pZERO vector. Sequencing reactions were performed with the ET sequencing terminator kit. Sequences were collected using a Megabase 1000 sequencer. SAGE tag sequences were extracted using SAGE 2000 software.

ORGANISM(S): Mus musculus

SUBMITTER: Sanggyu Lee

PROVIDER: E-GEOD-16139 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Publications

Detection of low-abundant novel transcripts in mouse hematopoietic stem cells.

Kim Hyeng-Soo HS Hwang Junmo J Kim Young-Hun YH Kim Seonggon S Lee Jae-Woong JW Kang Hyung-Sik HS Kim Kil Soo KS Ha Ji-Hong JH Chung Jin Woong JW Chang Kyu-Tae KT Ryoo Zae Young ZY Lee Sanggyu S

Molecular genetics and genomics : MGG 20090708 4

Gene expression profiles of hematopoietic stem cells (HSCs) provide clues for understanding molecular mechanisms of HSC behavior, including self-renewal and differentiation. We took advantage of serial analysis of gene expression (SAGE) to identify medium- and low-abundant transcripts expressed in HSCs/hematopoietic progenitor cells (HPCs). Among a total of 31,380 unique transcripts, 17,326 (55%) correspond to known genes and, 14,054 (45%) are low-copy transcripts that have no matches to current ...[more]

PMID: 19585147

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Project description:We performed a genome-wide analysis of gene expression in primary human CD151 myeloid progenitor cells. By using the serial analysis of gene expression (SAGE) technique, we obtained quantitative information for the expression of 37,519 unique SAGE-tag sequences. Of these unique tags, (i) 25% were detected at high and intermediate levels, whereas 75% were present as single copies, (ii) 53% of the tags matched known expressed sequences, 34% of which were matched to more than one known expressed sequence, and (iii) 47% of the tags had no matches and represent potentially novel genes. The correct genes were confirmed by application of the generation of longer cDNA fragments from SAGE tags for gene identification (GLGI) technique for high-copy tags with multiple matches. A set of genes known to be important in myeloid differentiation were expressed at various levels and used different spliced forms. This study provides a normal baseline for comparison of gene expression in myeloid diseases. The strategy of using SAGE and GLGI techniques in this study has broad applications to the genome-wide identification of expressed genes. (Lee S et al. The pattern of gene expression in human CD15+ myeloid progenitor cells. Proc Natl Acad Sci U S A. 98(6):3340-5, 2001; Lee S et al. Gene expression profiles in acute myeloid leukemia with common translocations using SAGE. Proc Natl Acad Sci U S A. 103(4):1030-5, 2006) Human bone-marrow mononuclear cells were isolated from bone marrow with FicollyPaque solution and stored in liquid nitrogen. Cells from three donors were thawed at 37°C, pooled, and used immediately for the isolation of myeloid progenitor cells with CD15 magnetic beads (Dynal, Oslo, Norway). The cells isolated by CD15 magnetic beads were lysed directly with TRIzol reagent for isolation of total RNA. mRNA was purified from 5 mg of total RNA with oligo(dT)25 beads. cDNA was synthesized with a cDNA synthesis kit, and SAGE was performed according to the SAGE protocol with the exceptions described in the original paper. SAGE-tag sequences were collected with the Big-Dye sequencing kit and ABI377 sequencer, and tag sequences were extracted with SAGE 300 software.

Dataset Information

Mus musculus hematopoietic stem cell

Publications

Detection of low-abundant novel transcripts in mouse hematopoietic stem cells.

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