## Description

This track displays aligned cDNA clone sequences from the Stem Cell Database (SCDb) to the mouse genome. SCDb is the work of the Lemischka and Moore labs (Princeton University) and the Stoeckert lab (University of Pennsylvania).

Multipotent hematopoietic stem cells (HSCs) are responsible for the production of at least eight distinct blood cell lineages. Regulatory mechanisms that are involved in determining the fate of stem cells are derived using molecular components of both stem cells and cells making up their environment. Elucidation of the pathways and networks that are involved requires the identification of these molecular components. This was achieved using high throughput DNA sequence analysis of both human and mouse cell lines. Clones were derived from subtracted cDNA libraries from HSCs and an HSC supportive stromal cell line for each species.

## Methods

In the investigation for HSCs, cDNA libraries from a highly purified HSC population and from a stem cell-depleted population were utilized (see Materials and Methods and Phillips et al., 2000). The HSC library was converted into single-stranded molecules, and DNA from the stem-cell-depleted library was used as a template to synthesize biotinylated RNA. Two successive rounds of hybridization of the HSC DNA and the non-stem-cell RNA were performed to remove DNA not specific to the HSCs. This subtraction step was followed by synthesis of the second strand for the remaining single-stranded stem cell DNA, and then several rounds of purification of the resulting DNA. Sequences present in a stem cell-depleted cell population were subtracted in this manner from the HSC libraries to remove "housekeeping" gene products and to enrich for transcripts produced by the HSCs. Randomly-selected clones were sequenced using a single-pass.

The sequences were then subjected to bioinformatics analyses, including queries of seven public databases: SwissProt, Genbank proteins and nucleotides, ESTs, murine and human EST contigs and SCDb itself. Additionally, ORF analysis included searches for protein motifs (Pfam, Prodom, SMART, Prosite, eMatrix), transmembrane helices (Tmpred) and signal peptides (SignalP). This information, along with that obtained from virtual Northerns, nearest neighbor analysis and PubMed queries, was used annotate the sequences. Northern blots, semi-quantitative PCR and microarray experiments were also performed. The resulting annotation may be found in the SCDb.

A similar method was used to investigate the transcripts expressed in the HSC supportive stromal cell line (Hackney et al., 2002). This track was assembled from blat alignments of SCDb as of 15 July, 2005, to the mouse genome (mm6/Mar. 2005) using default blat parameters.

For more information about the construction of the SCDb clone library and to access the freely available SCDb, see the SCDb home page. Computational methods have been employed to extensivly analyze these sequence sets. The preliminary results of the computational analyses and the raw sequences were previously available in the original Stem Cell Database (Phillips et al., 2000) and the Stromal Cell Database (StroCDB) (Hackney et al., 2002).

## References

Hackney, J.A., Charbord, P., Brunk, B.P., Stoeckert, C.J., Lemischka, I.R. and Moore, K.A. A molecular profile of a hematopoietic stem cell niche. Proc. Natl. Acad. Sci. USA 99(20), 13061-6 (2002).

Phillips, R.L., Ernst, R.E., Brunk, B., Ivanova, N., Mahan, M.A., Deanehan, J.K., Moore, K.A., Overton, G.C. and Lemischka I.R. The genetic program of hematopoietic stem cells. Science 288(5471), 1635-40 (2000).

Please see the SCDb Publications page for additional references.