LongSAGE profiling of nine human embryonic stem cell lines

Abstract

To facilitate discovery of novel human embryonic stem cell (ESC) transcripts, we generated 2.5 million LongSAGE tags from 9 human ESC lines. Analysis of this data revealed that ESCs express proportionately more RNA binding proteins compared with terminally differentiated cells, and identified novel ESC transcripts, at least one of which may represent a marker of the pluripotent state.

Figure 1

Pearson distance tree of human ESC libraries. ESC libraries do not cluster based on the genotype (compare WA01 and WA01-M), MEF feeder line (ES03 and ES04) or passage number (compare WA07 and WA01). Brain sub nig, LSAGE_Brain_normal_substantia_nigra_B_1; Breast epi, LSAGE_Breast_normal_myoepithelium_AP_IDC7; Pancreas, LSAGE_Pancreas_normal_B_1; Vascular endo; LSAGE_Vascular_endothelium_normal_liver_associated_AP_NLEC1.

Figure 2

Coverage of the MGC by ESC sequence tag-based transcriptomes. Unambiguous tags from published MPSS and short SAGE ESC transcriptomes were mapped to genes in the MGC and compared to identically treated LongSAGE transcriptomes. To assess the impact of tag number on gene identification, the proportion of MGC sequences detected was plotted against increasing numbers of tags. Coverage of the MGC increases with increased numbers of tags for ESC LongSAGE libraries even at levels above 200,000 tags. In contrast, coverage of the MGC by the MPSS library plateaus early with little increase in coverage observed with increased sampling depth (MPSS). Coverage of the MGC by the short SAGE ESC libraries (ES03_SAGE and ES04_SAGE) is significantly lower due to the presence of ambiguous tags.

Figure 3

Differentially expressed isoforms (as predicted by LongSAGE tag positions) for the Srfp1 transcript (see text). The tag sequence at position 9 results in the loss of the 3' UTR region targeted by evolutionarily conserved miRNAs. Putative miRNA target sites were predicted using miRanda [34] and are represented by hashed boxes.

Figure 4

Structure of the HA_003240 transcript. Alignment of the 5' RACE sequence for HA_003240 to chromosome 15 sequences, showing its position relative to the nested single exon transcript Foxb1 and conservation of the Octamer/Sox binding elements within the promoter region.

Figure 5

Structure of the Spd4 transcript. Alignment of the full-length Spd4 transcript on chromosome 3 showing its position relative to Dppa4 and conservation of the Octamer/Sox binding elements within the promoter region.

Figure 6

Expression of selected transcripts during embryoid body differentiation. qPCR was used to monitor expression of selected transcripts in ESCs stimulated to differentiate into embryoid bodies. Three control markers, Oct4, Lin28 and Msx1, were included. Expression levels are reported as the mean of triplicate measurements and are normalized to GAPDH.