Antisense Oligonucleotide Transfection and Analysis

Antisense moroholino oligonucleotide (AMO) transfection was performed by electroporation. The sequences of the U1 and control AMOs (Gene Tools) are 5′-GGTATCTCCCCTGCCAGGTAAGTAT-3′ and 5′-CCTCTTACCTCAGTTACAATTTATA-3′, respectively23 (link),24 (link). RNase H protection assay was carried out using AMO-transfected cell extracts and antisense DNA oligo for U1 snRNA (5′-CAGGTAAGTAT-3′). After RNase H treatment, RNA samples were purified and analyzed by Northern blotting with an U1 snRNA probe (5′-CAAATTATGCAGTCGAGTTTCCCACATTTG-3′). In situ hybridization of U1 snRNA was performed with a biotin-labeled LNA probe (5′-GGTATCTCCCCTGCCAGGTAAGTAT-3′). Nuclei were stained by DAPI. For in vitro splicing, [α–³²P] UTP labeled Ad2ΔIVS pre-mRNA was prepared as previously described40 (link). In vitro splicing reactions were carried out in 293T whole cell extracts prepared as previously described41 (link). Splicing products were resolved on denaturing PAGE, and gels were autoradiographed. For tiling array, labeled cDNA targets were prepared and applied to Affymetrix® GeneChip® Human tiling 2.0R E arrays. Arrays were scanned to produce .CEL files. The .CEL files were analyzed using the Affymetrix® Tiling Analysis Software (TAS) to produce .BED files of signal intensity and p-value. Overlapping regions of two datasets were chosen using Galaxy (http://galaxy.psu.edu/)42 (link). We produced .BAR files from the .CEL files using TAS to visualize on the Integrated Genome Browser (Affymetrix). For 3′ RACE, cDNA was synthesized from total RNA using an oligo dT18-XbaKpnBam primer. The first and second (nested) PCR reactions were performed using gene specific forward primers and the XbaKpnBam reverse primer. For 3′ RACE of NR3C1 mini-gene, pcDNA3.1-5′ primer was used as the first primer to distinguish mini-gene RNA from endogenous NR3C1 RNA. To construct the NR3C1 mini-gene, DNA fragments of NR3C1 intron 1-exon 2-intron 2 and NR3C1 intron 2-exon 3 were amplified and subcloned into pcDNA3.1 vector. The poly(A) site and 5′ splice site were mutated in this construct where indicated. Sequences of all primers are listed in Supplementary Table 1.