American Society of Hematology

Figure 2.

$Characterization of long noncoding RNA LOUP. (A) Gene track view of the genomic region encompassing the PU.1 locus. RNA-seq tracks include THP-1, HL60, primary monocytes, and Jurkat. DNase-seq and ChIP-seq are overlay tracks of monocyte and myeloid cell lines. These data were processed from published data in GEO (see “Methods” for details). The CAGE-seq track was imported from the FANTOM5 project; #1, #2, and arrows point to locations of the RNA peaks. (B) RT-PCR analysis of LOUP’s transcript features. First-strand cDNAs were generated from HL-60 total RNA using a primer that does not anneal to the PU.1 locus (Unrelated), random hexamers, Oligo(dT), and strand-specific primers (antisense and sense). (C) Northern blot analysis of LOUP. polyA− and polyA+ RNA fractions were isolated from U937 and Jurkat cells. Top panel: schematic of the probe location spanning exon junction (E1 and E2a; see supplemental Figure 2D). Middle panel: northern blot detection of LOUP’s major and minor transcripts. Bottom panel: RNA gel demonstrating relative migration between 28S and 18S rRNAs stained with ethidium bromide. (D) qRT-PCR analysis of LOUP levels in polyA− and polyA+ RNA fractions isolated from HL-60 cells. Error bars indicate SD (n = 3). ***P < .001. (E) Calculation of LOUP transcript per cell by qRT-PCR. The LOUP RNA standard curve was generated by in vitro transcription. Error bars indicate SD (n = 3). **P < .01; ****P < .0001. See also supplemental Figure 2 and supplemental Table 3.$

Characterization of long noncoding RNA LOUP. (A) Gene track view of the genomic region encompassing the PU.1 locus. RNA-seq tracks include THP-1, HL60, primary monocytes, and Jurkat. DNase-seq and ChIP-seq are overlay tracks of monocyte and myeloid cell lines. These data were processed from published data in GEO (see “Methods” for details). The CAGE-seq track was imported from the FANTOM5 project; #1, #2, and arrows point to locations of the RNA peaks. (B) RT-PCR analysis of LOUP’s transcript features. First-strand cDNAs were generated from HL-60 total RNA using a primer that does not anneal to the PU.1 locus (Unrelated), random hexamers, Oligo(dT), and strand-specific primers (antisense and sense). (C) Northern blot analysis of LOUP. polyA⁻ and polyA⁺ RNA fractions were isolated from U937 and Jurkat cells. Top panel: schematic of the probe location spanning exon junction (E1 and E2a; see supplemental Figure 2D). Middle panel: northern blot detection of LOUP’s major and minor transcripts. Bottom panel: RNA gel demonstrating relative migration between 28S and 18S rRNAs stained with ethidium bromide. (D) qRT-PCR analysis of LOUP levels in polyA⁻ and polyA⁺ RNA fractions isolated from HL-60 cells. Error bars indicate SD (n = 3). ***P < .001. (E) Calculation of LOUP transcript per cell by qRT-PCR. The LOUP RNA standard curve was generated by in vitro transcription. Error bars indicate SD (n = 3). **P < .01; ****P < .0001. See also supplemental Figure 2 and supplemental Table 3.

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