Project name: Mus musculus

Description: Identification of transcripts in different subpopulations of the HIV mouse ES cell line growing under self-renewing conditions (+Lif, +10FCS, GMEM media and plated on gelatin coated dishes). Subpopulations were identified and isolated based on the expression of the cell surface marker, SSEA 1 (a marker of murine ES cells) and expression of the venus protein, the cDNA of which was knocked into the Hex locus (Hhex). Following growth for 24 hours after initial plating, approximately 10,000,000 cells were dissasociated from dishes with the use of Cell Dissasociating Buffer (Gibco). Cells were resuspended in FACs buffer (10%FCS in PBS) and incubated on ice. Marking of SSEA 1expression was achieved by a 10 minute incubation with a mouse monoclonal antibody to SSEA 1 (MC-480, Developmental Hybridoma Studies Bank, University of Iowa). Following several washes with FACs buffer, cells were then incubated with an Alexa-647 conjugated anti-mouse IgM anitbody (Invitrogen) for a further 10 minutes. Cells were then washed and resuspended in FACs buffer and subpopulations were fractionated by flow cytometry from two different clones of the HIV cell line (clone 5.1 and clone 16.1). The aim of this array experimnt is to identify significant differences in transcript levels of the four subpopulation from the HIV cell line. Differences of transcript levels from the subpopulations should be consistant among the biological and technical replicates for each.Keywords: transcript identification designOverall design: Four subpopulations were collected from clones 5.1 and 16.1 of the HIV cell line as such: V-, S+; V+, S+; V-, S-; V-, S+, where V = Venus expression and S = SSEA 1 expression. RNA was extracted from each subpopulation of each fractionated clone and samples were supplied as duplicates. Therefore each subpopulation is represented as duplicates biologically and as duplicates technically (effectively four times for each). RNA isolation was performed with Trizol reagent according to the manufacturer's instructions. RNA pelletes were resuspended in Nuclease Free Water (ambion) and was measured using the Nanodrop system. 260/280 values for all samples were in the range of 1.8-2.0

Data type: Transcriptome or Gene expression

Sample scope: Multiisolate

Relevance: ModelOrganism

Last updated: 2008-11-05