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RT-PCR Primers

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									Supplemental Materials & Methods
RT-PCR primers
nPTB exon 8 forward: 5′-GCATTTGCCAAGGAGACATCC
nPTB exon 11 reverse: 5′-CGCTGCACATCTCCATAAACAC
PTB exon 8 forward: 5′-AAGAGCAGAGACTACACTCGA
PTB exon 12 reverse: 5′-CTGCCGTCTGCCATCTGCACAA
src N1 exon forward: 5′-GGCCCTCTATGACTATGAG
src N1 exon reverse: 5′-GCCAGCCACCAGTCTCCCTC
Smap1_2 exon forward: 5′-AGCAGTAGAGTTTTTTCATCAGAGAT
Smap1_2 exon reverse: 5′-GGCTTTTCTGGCTCCTTTTC
Mtap2_2 exon forward: 5′-TCCCCAGCTACTCCTAAGCA
Mtap2_2 exon reverse: 5′-GCCTGTGACGGATGTTCTTT
CLCB EN exon forward: 5′-GGAGGAGTGGAACCAGCGCC
CLCB EN exon reverse: 5′ –GGTCTCCTCCTTGGATTC
Kif1b_3 exon forward: 5′-CGGTTCCACTGGTTCAAACT
Kif1b_3 exon reverse: 5′-ACAGCCACACGAAGAAATCC
Dst_2 forward: 5′-GATCTCGGTGGTAGCTCAGG
Dst_2 reverse: 5′-TGTTTCCGGAGGAGAATGTC
Dst_3 forward: 5′-AAGCACAGTGATGGTTCGTG
Dst_3 reverse: 5′-TCCATGTTGGTCCTTCCTTT
AA536749_5 forward: 5′-TCTAAAAGCAATCCTGACTTCCTG
AA536749_5 reverse: 5′-GTGTCAGTGGAATGGATGCAGC
Mybl1_2 forward: 5′-CACTCTGCATCTGTGAAGAAG
Mybl1_2 reverse: 5′-TGTCTTCCCATATACCACTGTTTC
Chr5.809_1 forward: 5′-TGGCACCTCTAAAGAAAGCAA
Chr5.809_1 reverse: 5′-ACATGTTCCCCATGTCCAGT
Atp2b1_1 forward: 5′-GTGGCCAGATCTTGTGGTTT
Atp2b1_1 reverse: 5′-CATCAATAAGGGGGATGTGC
Ktn1_2 forward: 5′-TAGAGAAGGCCGAGATGGAG
Ktn1_2 reverse: 5′-CCTTCTTTCTCTCCGTTTGTTC

Real-time PCR primers
ACTB forward: 5′-TACAGCTTCACCACCACAGC
ACTB reverse: 5′-ATGCCACAGGATTCCATACC
GAPDH forward: 5′-
GAPDH reverse 5′-
PTB forward: 5′-TCTACCCAGTGACCCTGGAC
PTB reverse: 5′-GAGCTTGGAGAAGTCGATGC
nPTB forward: 5′-ACCAGGCATTTTTGGAACTG
nPTB reverse 5′-TGTGGTGCCACTAAGAGGTG

siRNA template oligonucleotides
pRL156(luciferase siRNA), sense: 5′-AATAAATAAGAAGAGGCCGCGCCTGTCTC
pRL156(luciferase siRNA), antisense: 5′-AACGCGGCCTCTTCTTATTTACCTGTCTC
Upf1 siRNA, sense: 5′-AATTCAGTTTTAGCAGTGGAACCTGTCTC
Upf1 siRNA, antisense: 5′-TTTTCCACTGCTAAAACTGAACCTGTCTC

shRNA construct oligonucleotides
si-m-PTB-forward
 5′-GAGAGAAGACTAAAAATGACTTACAGACCAGACATTATAAGAGTAA
si-m-PTB-reverse
 5′-GAGAGAAGACTAAAAATGACTTACAGACCAGAGATTACTCTTATAA
si-nPTB-v3-forward
 5′-GAGAGAAGACTAAAAATGGTCTAGATTCAGTTATGAATAAGAGTTC
si-nPTB-v3-reverse
 5′-GAGAGAAGACTAAAAATGGTCTAGATTCAGTTATGAACTCTTATTC

Construction of nPTB exon 10 minigenes

The primer nPTB-E10MG-F2A_Bgl was used for both minigenes B and C. The PCR

products were cloned using ApaI and BglII sites, replacing the middle exon of Dup4-1 in

which the constitutive exons and intervening intron sequences are derived from β-globin

(Modafferi, E.F. and D.L. Black. A complex intronic splicing enhancer from the c-src

pre-mRNA activates inclusion of a heterologous exon. (1997). Molec. and Cell. Biol.

17(11):6537-6545).

Minigene A:

nPTB_E10MG-F1_Apa: 5’-aaatgggcccATTCTGCGGGAACCACCCTTCGTTATG

nPTB_E10MG-R2_Bgl: 5’- aaaragatctGCCAAAGTGCTAGCTACAAATAAGAA

Minigene B:

nPTB-E10MG-F2A_Bgl:

5’- attagggcccATTTTCTGACCAAATTCCTGCATTTCCTATGTACTGACC

nPTB_E10MG-R1_Bgl: 5’- aaatagatctATAACATAccgaagagggtaaacaga

Minigene C:

nPTB-E10MG-F2A_Bgl:
5’-attagggcccATTTTCTGACCAAATTCCTGCATTTCCTATGTACTGACC

nPTB_E10MG-R3A_Bgl: 5’- Aaatagatctaaagaccatgcaaaataaattagcatgca


Splicing Microarray

Design and Construction of microarray

Exons for the array were selected based on the criteria presented in the Materials and

Methods section. The exon sets were combined, filtered for redundancies and used to

build models for the corresponding transcripts. Oligonucleotide probes for the exon-exon

junctions from skipped and included isoforms were designed, along with probes for

sequence within the alternative exon itself and within adjacent constitutive exons.

Oligonucleotides were spotted in duplicate. Briefly, 5' amino-linker containing

oligonucleotides at 33uM in print buffer (150 mM NaPO4, 6.25% Na2SO4, 0.0005%

SDS) were printed and covalently attached to Codelink slides (GE). Printing was done

on a linear servo-driven robotic contact pin printer essentially as described by the DeRisi

lab at UCSF (http://derisilab.ucsf.edu/arraymaker.shtml). Coupling and post-processing

of slides were described previously (Clark et al., 2002).

cDNA labeling and microarray hybridization

5 to 10µg of poly-A selected RNA were annealed to 450µmol mix of random hexamers

and dT VN. The RNA was then reverse transcribed in the presence of 300µM amino-
      24

allyl dUTP (Ambion), 100µM dTTP and 500µM each dATP, dCTP and dGTP. The

reaction was incubated for 2 hours at 48ºC and stopped by the addition of NaOH and

EDTA to final concentration of 100mM and 2mM respectively. The RNA was

hydrolyzed by incubation at 65ºC for 20 minutes and the solution was neutralized by the
addition of HEPES pH7.5 to 500µM final concentration. The cDNA was purified using

DNA clean and concentrator kit (Zymo research) and eluted in 50mM NaHCO buffer
                                                                       3

(pH9.0). The amino-allyl labeled cDNA was coupled to either Cy3 or Cy5 ester (GE)

according to the manufacturers protocol. The fluorescently labeled cDNA was purified

using DNA clean and concentrator kit and eluted in 14µl of 10mM Tris buffer pH8.0.

The hybridization mix was prepared by combining 10µl of Cy3 and Cy5 labeled samples

and adding 0.66µl 1M HEPES (pH7.5), 1.65µl poly-A (2µg/µl), 6.6µl 20xSSC and 3.3µl

1% SDS. The mix was denatured at 95ºC for 3 minutes, cooled down and applied to the

microarray slide. The hybridization was carried out at 63ºC for 16 to 20 hours. The slides

were then rinsed with 4XSSC/0.1% SDS prewarmed to 60C and washed twice for 5

minutes in 2xSSC/0.1% SDS prewarmed to 60ºC, and once in each 0.2xSSC and

0.05xSSC for 1 minute at room temperature. The slides were dried by centrifugation at

800rpm for 4 minutes and scanned immediately on a GenePix 4000b scanner (Molecular

Devices). During the prescan, the photomultipliers were adjusted to avoid saturation and

to balance the signal of the two channels.

Microarray image processing, array normalization and data visualization

Scanned image data were analyzed using GenePix Pro6 software (Axon) as described

previously (Srinivasan et al., 2005). Briefly, each spot was defined within a grid layout

over the image. Flagging criteria were applied to exclude bad spots as detailed

(Srinivasan et al., 2005). For each channel, the median values of the qualified spots were

determined after subtraction of local background intensities. The result files generated

from the GenePix pro 6 were then analyzed in R using Bioconductor. Spots with

intensities lower than the negative control spots were excluded from the analysis. Global
normalization was then performed using print-tip lowess method. For each gene, the

probes were categorized as constitutive, inclusion, or exclusion probes according to their

splicing models. To adjust for changes in overall expression, we performed “within-gene

normalization” by subtracting the average log2 ratio of the constitutive probes from the

log2 ratios of every individual alternative probe. To identify significant differences in

exon splicing, we performed two sample t-tests between the log2 ratios of the inclusion

probe set and the exclusion probe set, and calculated p-values for every alternative exon.

Alternative exons with a non-zero difference in log ratio between inclusion and exclusion

probes at a p-value of less than 0.05 were considered to have splicing changes.

Alternative exons with p-values lower than 0.05 were subjected to resampling-based false

discovery rate analysis (FDR), to control for the multiplicity and ranked by their FDR

value.

								
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