Project description: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.
Project description: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.
Project description:Cell surface markers of adult hematopoietic stem cells (HSCs) are well established, but fewer markers are available for embryonic HSCs and their immediate precursors (pre-HSCs) in the major arteries. We performed RNA-Seq on a population of cells from the major arteries of embryonic day 11.5 mouse embryos enriched for lymphoid progenitors, pre-HSCs, and HSCs. We determined that Tnfrsf7, encoding CD27, a member of the tumor necrosis factor receptor superfamily, is upregulated in this population. CD27 is found on ~5% of hematopoietic cluster cells in the major arteries, and enriches for progenitors with lymphoid potential, embryonic HSCs, and Type II pre-HSCs (CD144+CD45+), and fetal liver HSCs. It does not, however, enrich for yolk sac derived erythro-myeloid progenitors. Collectively, those data show that CD27 is a new cell surface marker of embryonic HSCs and Type II pre-HSCs.