Fig. 1

Analysis of HOXA5 transcripts using a next generation sequencing-based targeted RNA-capture system. a. After treating HCT116 cells with the indicated siRNAs for 48 h, HOXA5 mRNA levels were measured by qPCR, using GAPDH as an endogenous quantitative control. Data are expressed as the mean fold changes ± standard deviation (SD; n = 4), compared with those in control siRNA-treated cells. *Statistically significant difference versus control siRNA-treated cells (unpaired Student’s t-test, P < 0.01). b. The levels of HOXA5 protein were measured by western blotting. Coomassie brilliant blue (CBB) stain was used as a loading control. The amount of HOXA5 protein relative to that of CBB-stained bands was quantitatively analyzed by densitometry. c. After treating HCT116 cells with 10 nM of the indicated siRNAs, the cells were counted at the indicated times. Values are shown as the mean ± SD (n = 4). *Statistically significant difference versus control siRNA-treated cells (unpaired Student’s t-test, P < 0.01). d and e. HCT116 cells were treated following the rescue procedures described in the Methods section. HOXA5 protein levels in nuclear fractions were measured by western blotting (d). The cells were counted at the indicated times (e). The values shown represent the means ± SD (n = 4). *Statistically significantly difference versus control siRNA-treated and mock-transfected cells (unpaired Student’s t-test, P < 0.01). f. A DNA library was prepared using SMARTer Target RNA Capture with mixed probes a or b, as described in the Methods section. Sequence data from a MiSeq system were visualized using the Integrative Genomics Viewer. Sequence-read alignments from mixed probes A and B are indicated in red and blue, respectively. Grey boxes indicate HOXA5- and HOXA6-encoding sequences. Three transcripts (HOXA5 long 1, long 2, and short) are represented under the genome schematic based on the Target RNA Capture analysis, 5′-RACE, and 3′-RACE assays used to define their sequences