Efficient knock-in of DD-domain into the endogenous by TALENs in HCT116 cells PCR verification of Degron-KI clones Immunofluorescence staining of Degron-KI cells

Supplemental Table 3 page 1

List of alternatively spliced identified in response to SF3B1 mutant depletion using MATS geneSymbol event PValue FDR significant ABCC5 retained intron 0 0 yes ARMC9 alt. 3' splice site 0 0 yes DYNLL1 alt. 3' splice site 0 0 yes SNRPN skipped exon 0 0 yes TMEM14C alt. 3' splice site 0 0 yes mutually exclusive TMEM14C exon 0 0 yes SNRPN skipped exon 1.11E‐16 1.81E‐12 yes UBA1 skipped exon 8.88E‐16 9.66E‐12 yes ABCC5 alt. 3' splice site 1.19E‐13 2.39E‐10 yes SNRPN alt. 3' splice site 9.99E‐13 1.60E‐09 yes HSF4 alt. 3' splice site 4.96E‐12 6.62E‐09 yes HNRPDL retained intron 9.53E‐12 3.32E‐08 yes HSF4 retained intron 3.35E‐11 7.79E‐08 yes RBM18 alt. 3' splice site 1.47E‐10 1.68E‐07 yes HSF4 alt. 3' splice site 1.72E‐10 1.72E‐07 yes mutually exclusive TMEM14C exon 1.98E‐10 5.99E‐07 yes CHTF18 alt. 3' splice site 4.09E‐09 3.64E‐06 yes TYRP1 skipped exon 5.85E‐10 4.77E‐06 yes TTPA alt. 3' splice site 1.03E‐08 8.25E‐06 yes MFSD12 retained intron 5.01E‐09 8.74E‐06 yes MMP17 retained intron 7.02E‐09 9.79E‐06 yes ENOSF1 alt. 3' splice site 1.92E‐08 1.40E‐05 yes COPS2 alt. 3' splice site 2.45E‐08 1.64E‐05 yes BCL2L2 retained intron 2.07E‐08 2.41E‐05 yes mutually exclusive ST5 exon 2.10E‐08 4.23E‐05 yes C12orf32,RHNO1 alt. 3' splice site 7.89E‐08 4.86E‐05 yes IQGAP3 alt. 3' splice site 1.50E‐07 8.59E‐05 yes ANKHD1‐EIF4EBP3 alt. 3' splice site 1.64E‐07 8.77E‐05 yes NA alt. 3' splice site 2.83E‐07 0.0001415 yes mutually exclusive SNRPN exon 9.63E‐08 0.0001456 yes SERBP1 alt. 3' splice site 3.13E‐07 0.0001472 yes SCPEP1 retained intron 1.49E‐07 0.0001489 yes CCL28 alt. 3' splice site 5.43E‐07 0.0002071 yes ECT2 alt. 3' splice site 5.16E‐07 0.0002071 yes HTR2B alt. 3' splice site 5.12E‐07 0.0002071 yes Supplemental Table 3 page 2

INTS9 alt. 3' splice site 5.18E‐07 0.0002071 yes CCL28 alt. 5' splice site 5.45E‐08 0.0003846 yes mutually exclusive ST5 exon 3.33E‐07 0.0004025 yes RHOBTB3 alt. 3' splice site 1.67E‐06 0.0006077 yes GANC retained intron 7.43E‐07 0.0006479 yes TRIP12 alt. 3' splice site 2.05E‐06 0.0007136 yes MBTPS2 alt. 3' splice site 4.33E‐06 0.0014428 yes HMGN3 retained intron 1.91E‐06 0.0014819 yes TEX30 alt. 3' splice site 5.32E‐06 0.001704 yes MBTPS2 alt. 3' splice site 5.73E‐06 0.0017636 yes RPRD1A retained intron 2.87E‐06 0.0020033 yes PPP2R3A alt. 3' splice site 7.64E‐06 0.002266 yes NACC2 alt. 3' splice site 1.31E‐05 0.0036241 yes ZDHHC16 alt. 3' splice site 1.27E‐05 0.0036241 yes SPAG7 alt. 3' splice site 1.40E‐05 0.0037453 yes DLST alt. 3' splice site 1.60E‐05 0.0041362 yes SPARC alt. 5' splice site 1.22E‐06 0.0043011 yes GCC2 alt. 3' splice site 1.75E‐05 0.0043855 yes SEPT2 alt. 3' splice site 1.84E‐05 0.0044678 yes INTS9 retained intron 8.09E‐06 0.0051302 yes EIF4E2 retained intron 8.89E‐06 0.0051716 yes NA alt. 3' splice site 2.27E‐05 0.0051859 yes SUGP1 alt. 3' splice site 2.23E‐05 0.0051859 yes DDX39B retained intron 1.00E‐05 0.0052509 yes LY6G5B retained intron 1.09E‐05 0.0052509 yes PKM2,PKM retained intron 1.13E‐05 0.0052509 yes PRMT7 retained intron 1.29E‐05 0.0056054 yes TNFRSF14 retained intron 1.62E‐05 0.0066632 yes TMEM136 alt. 3' splice site 3.06E‐05 0.0067976 yes TRIM37 alt. 3' splice site 3.26E‐05 0.0070443 yes SLC37A1 skipped exon 1.43E‐06 0.0093143 yes LUC7L3 retained intron 2.83E‐05 0.0109841 yes PILRB retained intron 3.04E‐05 0.0111511 yes NSMCE4A alt. 3' splice site 5.59E‐05 0.0114637 yes PHGDH alt. 3' splice site 5.55E‐05 0.0114637 yes ASXL1 retained intron 3.41E‐05 0.0119102 yes AP4M1 retained intron 3.61E‐05 0.011987 yes HADHA alt. 3' splice site 6.19E‐05 0.0123943 yes NCDN retained intron 4.00E‐05 0.0126735 yes RARG mutually exclusive 1.59E‐05 0.0152879 yes Supplemental Table 3 page 3

exon mutually exclusive SNRPN exon 1.77E‐05 0.0152879 yes FAM65B alt. 3' splice site 8.11E‐05 0.0158367 yes LHFPL3‐AS1,LOC645591 alt. 3' splice site 8.37E‐05 0.0159594 yes TMEM136 alt. 3' splice site 9.17E‐05 0.017075 yes POLM alt. 3' splice site 0.0001004 0.0182589 yes TNPO3 alt. 3' splice site 0.0001129 0.02009 yes BNIP3 alt. 3' splice site 0.0001193 0.020581 yes PLXNA3 alt. 3' splice site 0.0001209 0.020581 yes ZMYM4 alt. 3' splice site 0.0001234 0.020581 yes MEGF6 alt. 3' splice site 0.0001274 0.020695 yes NPLOC4 alt. 3' splice site 0.0001293 0.020695 yes CHTF18 retained intron 7.33E‐05 0.0222364 yes MCAM retained intron 7.71E‐05 0.0224248 yes MICAL1 alt. 3' splice site 0.0001476 0.0231592 yes ANKHD1‐EIF4EBP3 alt. 3' splice site 0.0001518 0.0233611 yes BNIP3 retained intron 9.13E‐05 0.0236856 yes SRSF11 retained intron 8.59E‐05 0.0236856 yes ZWINT retained intron 9.17E‐05 0.0236856 yes SMARCD3 alt. 3' splice site 0.0001573 0.0237541 yes IL17RC retained intron 9.62E‐05 0.0239832 yes mutually exclusive TTPA exon 3.24E‐05 0.0245137 yes METTL22 alt. 3' splice site 0.0001667 0.0247106 yes GCLC alt. 3' splice site 0.0001747 0.0254163 yes ZDHHC16 alt. 3' splice site 0.0001811 0.0258788 yes MMP17 skipped exon 4.97E‐06 0.0270258 yes AURKB alt. 3' splice site 0.0001966 0.0276054 yes PLEKHA5 alt. 3' splice site 0.0002013 0.0277753 yes MEGF6 alt. 3' splice site 0.000209 0.0283527 yes ERLIN1 retained intron 0.0001223 0.0287716 yes SRCAP retained intron 0.0001237 0.0287716 yes ANAPC5 alt. 3' splice site 0.0002237 0.0293537 yes LOC100289561 alt. 3' splice site 0.000223 0.0293537 yes PDIA3P alt. 3' splice site 0.000233 0.0296048 yes PKP4 alt. 3' splice site 0.0002315 0.0296048 yes GTF2I alt. 3' splice site 0.0002552 0.0319134 yes C1orf159 alt. 3' splice site 0.0002593 0.0319244 yes mutually exclusive C11orf74 exon 5.28E‐05 0.0321637 yes Supplemental Table 3 page 4

mutually exclusive SNRPN exon 5.32E‐05 0.0321637 yes CCDC74A retained intron 0.0001463 0.0329253 yes STIM1 alt. 3' splice site 0.0002779 0.0336978 yes HLA‐G alt. 3' splice site 0.0002876 0.0338182 yes PDIA3P alt. 3' splice site 0.0002884 0.0338182 yes ZNF41 alt. 3' splice site 0.0002915 0.0338182 yes mutually exclusive HKR1 exon 7.12E‐05 0.0347226 yes mutually exclusive IMMP1L exon 7.46E‐05 0.0347226 yes mutually exclusive PI4KB exon 6.33E‐05 0.0347226 yes mutually exclusive C17orf53 exon 8.31E‐05 0.0358921 yes ARMC8 alt. 3' splice site 0.0003332 0.0375618 yes SLC3A2 alt. 3' splice site 0.0003311 0.0375618 yes GANC retained intron 0.0001913 0.0404491 yes SMARCD3 retained intron 0.0001912 0.0404491 yes C12orf76 alt. 3' splice site 0.0003894 0.0421232 yes MAP3K7 alt. 3' splice site 0.0003805 0.0421232 yes PPP2R5A alt. 3' splice site 0.0003888 0.0421232 yes MEGF6 alt. 3' splice site 0.0004004 0.042728 yes FAM211B alt. 3' splice site 0.0004305 0.0453426 yes AFG3L1P alt. 3' splice site 0.0004445 0.0461004 yes ATP8B2 alt. 3' splice site 0.0004597 0.0461004 yes C1orf51 alt. 3' splice site 0.0004538 0.0461004 yes TIAL1 alt. 3' splice site 0.0004608 0.0461004 yes ECHDC2 alt. 3' splice site 0.0004727 0.0467061 yes HDAC6 retained intron 0.0002351 0.0481112 yes RANGAP1 retained intron 0.0002413 0.0481112 yes

Supplemental Table 4 page 1 Comparison of the alternatively spliced genes identified in this study with genes reported in prior reports

p- method p-value p-adjusted significant adjusted MATS ABCC5 0.0000 0.0000 yes 0.0000 MATS ACTR1B 0.0002 0.0806 no 1.0000 MATS CRNDE 0.0000 0.0000 no 1.0000 MATS DHPS 0.0000 0.0005 no 0.8450 MATS EI24 0.0000 0.0000 no 0.8992 MATS ERGIC3 0.0000 0.0000 no 0.7831 MATS GANAB 0.0000 0.0110 no 0.8571 MATS GPR108 0.0000 0.0000 no 0.6238 MATS HAX1 0.0000 0.0005 no 0.8076 MATS MCM7 0.0000 0.0000 no 1.0000 MATS MOV10 0.0001 0.0691 no 0.0749 MATS NDUFS5 0.0000 0.0559 no 1.0000 MATS PSME2 0.0000 0.0128 no 1.0000 MATS QARS 0.0001 0.0281 no 0.8503 MATS QTRT1 0.0001 0.0367 no N/A MATS RABGGTA 0.0000 0.0134 no 0.7139 MATS RPS6KB2 0.0001 0.0527 no 1.0000 MATS RPS9 0.0000 0.0017 no 0.9492 MATS SELENBP1 0.0000 0.0128 no 0.8094 MATS SERBP1 0.0001 0.0614 yes 0.0001 MATS SNRPN 0.0000 0.0000 yes 0.0000 MATS THRA 0.0000 0.0100 no 1.0000 MATS TMEM214 0.0000 0.0000 no 0.7463 MATS WSB1 0.0000 0.0000 no 0.6412 MATS ZNF142 0.0000 0.0005 no 0.5719

p- method gene p-value p-adjusted significant adjusted DEXSeq ABCC5 0.0000 0.0206 yes 0.0000 C17orf76- DEXSeq AS1 0.0000 0.0156 no 0.0751 DEXSeq CRNDE 0.0000 0.0022 no 1.0000 DEXSeq DLST 0.0000 0.0069 yes 0.0041 DEXSeq DYNLL1 0.0000 0.0021 yes 0.0000 DEXSeq EI24 0.0000 0.0000 no 0.8992 DEXSeq ENOSF1 0.0000 0.0026 yes 0.0000 DEXSeq ERGIC3 0.0000 0.0001 no 0.7831 DEXSeq FHIT 0.0000 0.0026 no 0.8795 DEXSeq MAD1L1 0.0000 0.0040 no 0.4609 Supplemental Table 4 page 2 Comparison of the alternatively spliced genes identified in this study with genes reported in prior reports

DEXSeq RPL27A 0.0000 0.0000 no 1.0000 DEXSeq RPL31 0.0000 0.0111 no 1.0000 DEXSeq SCIN 0.0000 0.0253 no N/A DEXSeq SLC3A2 0.0000 0.0007 yes 0.0376 DEXSeq STIM1 0.0000 0.0206 yes 0.0337 DEXSeq SVIP 0.0000 0.0796 no N/A DEXSeq TCEA2 0.0000 0.0069 no 0.1896 DEXSeq TMEM14C 0.0000 0.0000 yes 0.0000 DEXSeq TRIM33 0.0000 0.0279 no 1.0000 DEXSeq UQCC 0.0000 0.0000 no 0.7658

Supplemental Figure 1. Efficient knock‐in of DD‐domain into the endogenous p53 locus by TALENs in

HCT116 cells

(A) 24 randomly chosen clones were cultured in the presence of 1uM Shld and collected for western blot. DD tagged p53 runs at a higher molecular weight due to the addition of 12kd DD tag. DD‐p53 marks the DD‐tagged allele, whereas p53 indicates the migration of untagged p53. GAPDH serves as a loading control. Note that some of the clones have both the endogenous and the DD‐p53 band (heterozygous

DD targeting), while some of the clones only have the DD‐p53 band (homozygous DD targeting or DD‐ tagged over a null allele).

Supplemental Figure 2. PCR verification of Degron‐KI clones

(A‐E) Schematic depiction of PCR strategy to distinguish homozygous DD tagged clones from heterozygous DD tagged clones. The forward and reverse PCR primers are within the homologous recombination arms (black regions). If all the alleles are tagged with DD, the endogenous lower PCR band should be absent due to the insertion of the PuroR‐2A‐DD sequence. Representative clones for

Degron‐KI engineered ESS‐1 and Mel202 are shown. The lower PCR band is sequenced to assess the status of the untagged allele, which can be either wild‐type or subject to insertion/deletion due to error prone repair by NHEJ.

(F‐J) Junction PCR to confirm the insertion of DD into the endogenous locus. The forward primer is within the DD domain, while the reverse primer is outside the right homologous recombination arm.

Therefore, the PCR only produces a correctly sized product when the DD tag is inserted into the endogenous locus.

Supplemental Figure 3. Immunofluorescence staining of Degron‐KI cells

(A‐B) Degron‐KI cells and their parental cells were cultured with/without Shld for three days before immunofluorescence staining. Note that p53 staining (A) was not uniform among cells. Shld withdrawal led to a marked reduction of IF intensity in both p53 and EZH2 Degron‐KI lines but not in the parental lines. DAPI staining was used to mark the nucleus. DD‐tagging and Shld withdrawal did not seem to affect the nucleus localization of the DD‐tagged . Representative pictures are shown.

Supplemental Figure 4. Quantification of DD tagged proteins in the presence or absence of Shld.

(A‐F) Quantification of levels in western blots shown in the main figures; the western blots are duplicates of the blots in the main figures to allow for easy correlation with the protein quantification. Protein quantification was conducted using Image J, and the remaining DD tagged protein levels after Shld withdrawal was normalized to the protein levels before Shld withdrawal for each clone (see details in methods). It is important to note that for some proteins, the DD‐tagged protein levels in the presence of Shld appeared to be slightly lower compared to the endogenous untagged allele. Thus, the amount of protein depletion (compared to untagged endogenous protein) is likely to be more complete than estimated by this ‘relative’ (+/‐Shld) quantification.

Supplemental Figure 5. Highly efficient Degron‐KI at the endogenous EZH2 locus by CRISPR in HCT116

(A) EZH2 donor vector together with either CRISPR Cas9 or Cas9 D10 nickase mutant were transfected into HCT116 cells. After puro selection, pooled surviving cells were collected for western blot. Note that the use of Cas9 wild‐type enzyme mediated Degron‐KI is so efficient in HCT116 that the endogenous lower EZH2 band is almost absent in the pooled (polyclonal) population.

(B) Use of Cas9 nickase (D10) mutant enzyme yields efficient heterozygous Degron‐KI. 24 individual clones from the Cas9 D10 pool were treated with 1uM Shld and collected for western blot. Every clone has heterozygous DD knock‐in. Note that only one Cas9 D10 arm was used here instead of a pair

(expected to produce nick‐ase activity rather than double stranded breaks).

(C) HCT116 DD‐EZH2/‐ #1 and #2 have DD inserted into one EZH2 allele, while the other allele undergoes repair by error‐prone NHEJ, resulting in small deletions (sequences of NHEJ alleles are shown here). These deletions cause a reading frame shift around the start codon (ATG) so that the NHEJ allele is not expressed, or is expressed as an aberrant truncated protein.

Supplemental Figure 6. Assessment of the kinetics of DD‐EZH2 and DD‐SF3B1 depletion upon Shld withdrawal

(A,B) Lysates were harvested at the indicated times post‐Shld withdrawal and blotted with the indicated antibodies.

Supplemental Figure 7. HCT116 (EZH2 wild type) cells are not sensitive to EZH2 inhibitor EI1

(A,B) EI1 (5uM) treatment significantly reduced H3K27Me3 in HCT116(A), but did not impair cell proliferation in the colony formation assay(B). EI1 has been demonstrated as a potent EZH2 inhibitor and has strong anti‐proliferation activities against lines harboring EZH2 gain of function mutations (1).

Supplemental Figure 8. RT‐PCR strategy to identify allele‐ specific DD tagging of mutant versus wild‐ type SF3B1

(A,B) Schematic depiction of RT‐PCR strategy for the DD tagged allele. The forward primer is within the

DD region, while the reverse primer is downstream of the SF3B1 hotspot mutation. Nested PCR is conducted to further increase the PCR signal. PCR products are analyzed by Sanger Sequencing to identify mutations. Representative clones are shown. ESS‐1 DD‐SF3B1 clone#3 is a heterozygous DD knock‐in clone.

(C,D) Schematic depiction of RT‐PCR strategy for the untagged allele. The forward primer spans across the 5’UTR and the starting coding sequence. DD‐tag insertions will destroy the forward primer annealing sequence. Therefore, this RT‐PCR will only amplify the untagged allele. ESS‐1 DD‐SF3B1#2 has all the alleles tagged with DD, while ESS‐1 DD‐SF3B1#3 is a heterozygous DD knock‐in clone (Supplemental

Figure 2E). Therefore, RT‐PCR works for clone #3 but not for #2. Note that some clones will undergo NHEJ for the untagged allele. The forward PCR primer may tolerate changes of a few nucleotides. But if the mismatches are too significant, one needs to design a specific forward primer for the specific untagged allele. When sequencing ESS‐1 cells, we noted that this cell line harbors an additional single nucleotide variant (R315Q) in SF3B1, which has not been reported in primary tumors and we suspect to be a passenger mutation. The R315Q and K666N mutations of SF3B1 in ESS‐1 always co‐occur in the RT‐

PCR products in multiple clones. Therefore, we conclude that these two mutations are on the same allele. For more than 10 clones of ESS‐1 or Mel202 Degron‐KI lines that were seqeuenced, whenever the

DD tagged allele RT‐PCR product is mutant, the untagged allele is exclusively wild type, suggesting that there is only one mutant SF3B1 allele in both cell lines.

Supplemental Figure 9. UQCC and CRNDE exhibit an altered splice pattern in SF3B1 mutant uveal melanoma cell lines

(A) UQCC: SF3B1 wild‐type cells use the last three exons (red boxed) as terminal exons while the SF3B1 mutant cell line Mel202 uses the previous exon as the terminal exon. CRNDE: SF3B1 mutant cell line

Mel202 uses an alternative 3’ splicing acceptor within exon 4 (red boxed).

Supplemental Figure 10. Shld has minimal effects on of parental Mel202 cells and

Mel202 DD‐mut‐SF3B1.

(A) Rescaled FPKM values are plotted on a log‐log scale for individual replicates of the Mel202 parental cell line without Shld (Y‐axis) and with Shld (X‐axis, 4 day treatment). A representative of 4 replicates is shown, with the other three replicates exhibiting a similar pattern. Among all pairwise comparisons of expression values from parental cell lines in the presence or absence of Shld, no genes were reported as significantly differentially expressed by Cuffdiff (2) using quartile normalization.

(B) The same analysis was performed on Mel202 DD‐SF3B1#2 (DD‐mut‐SF3B1, 2 days after Shld withdrawal). Using the methods described above, no genes were found to be significantly differentially expressed. Supplemental Figure 11. Selective depletion of mutant SF3B1 in Mel202 Degron‐KI cells reversed the pattern of DYNLL1, SNRPN, TMEM14C, ABCC5, ZDHHC16, RBM18.

(A) Visual plots of alternative splicing pattern of six candidate genes based on RNA‐seq data. DNYLL1,

SNRPN, TMEM14C, ABCC5 were identified in both this study and previous reports. ZDHHC16 and RBM18 were identified as alternatively spliced in SF3B1 mutants only in this study. Note that for these six genes, Mel202 parental cells have a different splicing pattern compared to Mel202 mut‐SF3B1 KO

(Mel202 DD‐SF3B1 #3) cells. Selective depletion of mutant SF3B1 in mel202 Degron‐KI cells (Mel202 DD‐

SF3B1 #2) reversed the splicing pattern. Samples were collected after 2 days of Shld withdrawal.

Supplemental Figure 12. Selective depletion of mutant SF3B1 in Mel202 Degron‐KI cells by Shld withdrawal predominantly alters 3’ splice sites but not 5’ splice site selection.

(A) Graph displays the differential splicing events upon Shld withdrawal in mutant‐SF3B1 Degron‐KI cells

(top panel) and parental cell line (bottom panel). Summary of differential splicing events were reported by rMATS. X‐axis displays the percent difference of relative inclusion of the respective exon or junction in the absence compared to presence of Shld. The Y‐axis represent the statistical significance of the differential splicing event (‐log10 of p‐value). Only splicing events that result in robust (at least a 25% change) in alternative splicing are shown. Samples were collected after two days of Shld withdrawal.

Supplemental Figure 13. Confirmation that the DD tag insertion occurred exclusively at the SF3B1 locus in Degron‐KI engineered Mel202 clones

(A) DD tag is a derivative of FKBP1A. DD (FKBP1A)‐SF3B1 fusions in indicated Mel202 DD‐SF3B1 clones were detected from discordantly aligned RNA‐seq read pairs and plotted using Circos (3). For simplicity, only fusions involving DD (FKBP1A) are shown. All DD (FKBP1A) fusions are with SF3B1 and are in frame.

Supplemental Table 1: Detailed genotypes of ESS‐1 Degron‐KI clones

There are four groups of ESS‐1 Degron‐KI clones. Group 1 (clone #1 and #2): Shld withdrawal depletes all

SF3B1. Clone #1 has one untagged allele with a large deletion (Supplemental Figure 2E). Group 2 (clone

#3 and #4): Shld withdrawal depletes only the mutant SF3B1 protein. Group 3 (clone #5 and #6): Shld withdrawal depletes mutant and some of the wt SF3B1 protein, but some untagged wt SF3B1 protein is still expressed. Group 4(clone #7, #8 and #9): Shld withdrawal depletes all wt SF3B1 protein, only untagged mutant SF3B1 is expressed. ATG is the starting codon. Note that in clone #8, the untagged mutant allele has a deletion of “A” before ATG; in clone #9, the untagged mutant allele has an in‐frame deletion of “GCG” after ATG.

Supplemental Table 2: Detailed genotypes of Mel202 Degron‐KI clones

There are three groups of Mel202 Degron‐KI clones. Group 1 (clone #1 and #2): Shld withdrawal depletes only mutant SF3B1. Group 2 (clone #3): Shld withdrawal depletes all SF3B1. Note that clone #3 is a SF3B1 mutant KO cell line. The untagged mutant allele has an indel due to NHEJ (the sequence around starting codon ATG changed from ACAAAATGGCG to A‐‐‐‐‐‐‐GTT; loss of starting codon ATG); but all wt alleles are tagged with DD. Group 3 (clone #4): Shld withdrawal depletes all wt SF3B1, but the untagged mutant SF3B1 is still expressed (the mutant allele has a deletion of “ACAAA” before ATG which appears to have little to no impact on expression).

Supplemental Table 3: List of alternatively spliced genes identified in response to SF3B1 mutant depletion using MATS.

RNA‐Seq data was analyzed using MATS (4) and all genes that showed significantly altered splicing

(p>0.05) are listed. Samples of Mel202 DD‐mut‐SF3B1 (Mel202 DD‐SF3B1 #2) cells were collected and subjected to RNA‐Seq after two days of Shld withdrawal. Detailed information was provided in the Excel file. Supplemental Table 4: Comparison of the alternatively spliced genes identified in this study with genes reported in prior reports (5).

The list of alternative splicing genes that were reported previously (5). The genes highlighted in yellow were also identified in this study. Note that CRNDE and UQCC were not identified using MATS in this study, but both genes clearly showed altered splicing patterns upon visual inspection of the RNAseq data, as shown in Figure 5C. This is probably due to the low expression levels of CRNDE and UQCC in the samples used in our study that likely caused MATS to miss these two genes, illustrating some of the limitations of current algorithms used to detect alternative splicing events.

Supplemental References:

1. Qi W, Chan H, Teng L, Li L, Chuai S, Zhang R, et al. Selective inhibition of Ezh2 by a small molecule inhibitor blocks tumor cells proliferation. Proc Natl Acad Sci U S A 2012;109(52):21360‐ 5. 2. Trapnell C, Williams BA, Pertea G, Mortazavi A, Kwan G, van Baren MJ, et al. Transcript assembly and quantification by RNA‐Seq reveals unannotated transcripts and isoform switching during cell differentiation. Nat Biotechnol 2010;28(5):511‐5. 3. Krzywinski M, Schein J, Birol I, Connors J, Gascoyne R, Horsman D, et al. Circos: an information aesthetic for comparative genomics. Genome Res 2009;19(9):1639‐45. 4. Park JW, Tokheim C, Shen S, Xing Y. Identifying differential alternative splicing events from RNA sequencing data using RNASeq‐MATS. Methods Mol Biol. 2013;1038:171‐9. 5. Furney SJ, Pedersen M, Gentien D, Dumont AG, Rapinat A, Desjardins L, et al. SF3B1 mutations are associated with alternative splicing in uveal melanoma. Cancer Discov 2013;3(10):1122‐9.

Supplemental Experimental Procedures:

Donor Vector:

Left arm puro P2A DD Ecor1 Right arm

ATGAC

CGAGTACAAG CCCACGGTGC GCCTCGCCAC CCGCGACGAC GTCCCCCGGG CCGTACGCAC

CCTCGCCGCC GCGTTCGCCG ACTACCCCGC CACGCGCCAC ACCGTCGACC CGGACCGCCA

CATCGAGCGG GTCACCGAGC TGCAAGAACT CTTCCTCACG CGCGTCGGGC TCGACATCGG

CAAGGTGTGG GTCGCGGACG ACGGCGCCGC GGTGGCGGTC TGGACCACGC CGGAGAGCGT

CGAAGCGGGG GCGGTGTTCG CCGAGATCGG CCCGCGCATG GCCGAGTTGA GCGGTTCCCG

GCTGGCCGCG CAGCAACAGA TGGAAGGCCT CCTGGCGCCG CACCGGCCCA AGGAGCCCGC

GTGGTTCCTG GCCACCGTCG GCGTCTCGCC CGACCACCAG GGCAAGGGTC TGGGCAGCGC

CGTCGTGCTC CCCGGAGTGG AGGCGGCCGA GCGCGCCGGG GTGCCCGCCT TCCTGGAGAC

CTCCGCGCCC CGCAACCTCC CCTTCTACGA GCGGCTCGGC TTCACCGTCA CCGCCGACGT

CGAGGTGCCC GAAGGACCGC GCACCTGGTG CATGACCCGC AAGCCCGGTG CC

GGA AGC GGA GCT ACT AAC TTC AGC CTG CTG AAG CAG GCT GGA GAC GTG GAG GAG AAC CCT GGA CCT

ATG GGAGTGCAGG

TGGAAACCAT CTCCCCAGGA GACGGGCGCA CCTTCCCCAA GCGCGGCCAG ACCTGTGTGG

TGCACTACAC CGGGATGCTT GAAGATGGAA AGAAAGTCGA TTCCTCCCGG GACAGAAACA

AGCCCTTTAA GTTTATGCTA GGCAAGCAGG AGGTGATCCG AGGCTGGGAA GAAGGGGTTG

CCCAGATGAG TGTGGGTCAG AGAGCCAAAC TGACTATATC TCCAGATTAT GCCTATGGTG

CCACTGGGCA CCCAGGCATC ATCCCACCAC ATGCCACTCT CGTCTTCGAT GTGGAGCTTC

TAAAACCGGA ACCG

GAATTC

Donor vector map

Below are the primers used to PCR out the left and right homologous arms from the genomic DNA.

P53:

Left arm:

F: TAAGAGATGCATATGATGAGATTAAAGAAGCCGAGACGGGC

R: CTTGTACTCCTCGAGGGCAGTGACCCGGAAGGCAGTCTGG

Right arm:

F: CCGGAACCGGAATTC ATGGAGGAGCCGCAGTCAGATCCTAG

R: TTTTCAAAAGCGGCCGCTCTTTGAGAGTGCTGGGATTGCAG

PIK3CA:

Left arm:

F: TAAGAGATGCATATGCATCTTCTAAAAGACCCCCCAAGATTG

R: CTTGTACTCCTCGAGATGGTCGTGGAGGCATTGTTCTGATTC

Right arm:

F: CCGGAACCGGAATTC CCTCCACGACCATCATCAGGTGAACTG

R: TTTTCAAAAGCGGCCGCGAGGTCCCTAAGATCCACAGCTTCTTTACAAACG

EZH2:

Left arm:

F: TAAGAGATGCATATGCCTGGTTGCAGTTATTCTTTCACTCC

R: CTTGTACTCCTCGAGGATTATTCTAAAAGCAATGGTTTCATATTAAAATCAC

Right arm:

F: CCGGAACCGGAATTCGGCCAGACTGGGAAGAAATCTGAG

R: TTTTCAAAAGCGGCCGCATGGACACCCTGAGGTCAATGATTTC SF3B1:

Left arm:

F: TAAGAGATGCATATGGCTGCTTCCGGGACCCATCTTTC

R: CTTGTACTCCTCGAGTTTGTCCACTCGAACACACAGACGGAACT

Right arm:

F: CCGGAACCGGAATTCATGGCGAAGATCGCCAAGACTCACGAAG

R: TTTTCAAAAGCGGCCGCGTCTCTTACCTCTCACAGACTAATACCGAGCC

Junction PCR: Forward primer against DD, reverse primer against a region that is outside of the HR arm.

Locus PCR: Both forward and reverse primers are within the HR arms.

P53 primers:

Junction pcr:

Forward: ATGCCACTCTCGTCTTCGATGTGGAGC

Reverse: CTCCGTCATGTGCTGTGACTGCTTG

P53 locus pcr:

Forward: GGGAGTTGGGAATAGGGTGCACATTTAGG

Reverse: CGGGGACAGCATCAAATCATCCATTGCTTG

EZH2 primers:

Junction pcr:

Forward: ATGCCACTCTCGTCTTCGATGTGGAGC

Reverse: GTGTGATCTACAGCAGTCATTAACAGTTGCAC

EZH2 locus pcr:

Forward: GACATCTGGTGAACTATAAGCTGTCCCC

Reverse: ATGGACACCCTGAGGTCAATGATTTC

PIK3CA primers:

Junction pcr:

Forward: ATGCCACTCTCGTCTTCGATGTGGAGC

Reverse: GACATACATTGCTCTACTATGAGGTGAATTGAGGTCCC

PIK3CA locus pcr: Forward: CCCATAGCCTCATCTCTGTCTTGATGAAACC

Reverse: ACGGTTGCCTACTGGTTCAATTACTTTTAAAAAGGG

SF3B1 primers:

Junction pcr:

Forward: ATGCCACTCTCGTCTTCGATGTGGAGC

Reverse: CATGCATCTGGCCTCATGAGAGAATGAG

SF3B1 locus pcr:

Forward: CGTAGAATCCACTTTCCTCCAGGGCAAAG

Reverse: CCAAACACTGAACAAAGCGCCTGTCG

SF3B1 RT-PCR primers:

Nested pcr primers to RT-PCR the DD-SF3B1 fusion cDNA region:

Primer set1:

Forward: CCCAGATGAGTGTGGGTCAGAGAGC

Reverse: TCTGTGTTGGCGGATACCCTTCC

Primer set2:

Forward: ATGCCACTCTCGTCTTCGATGTGGAGC

Reverse: GAGTTGCTGCTTCAGCCAAGGC

PCR primers to RT-PCR the untagged SF3B1 cDNA region:

Forward: GTGTTCGAGTGGACAAAATGGCGAAGATC

Reverse: GTAGTTTGCTTCTACACCATCTGTCCCAC

Please note that a few nucleotides need to be changed in the forward primer if there is non-homologous end joining in the untagged endogenous locus (if the deletion or mismatches are too significant) TALENs and CRISPR targeting sequences (ATG is the starting codon; underlying sequences are where TALENs and CRISPR target):

TALENs targeting p53 loci:

GATCCCCACTTTTCCTCTTGCAGCAGCCAGACTGCCTTCCGGGTCACTGC

CATGGAGGAGCCGCAGTCAGATCCTAGCGTCGAGCCCCCTCTGAGTCAGG

TALENs targeting SF3B1 loci:

GCGCAGAGTGCAGCCCCCAGCTATTTTTCTCCGTGGCGGCGGCGACGAGCGGAA GTTCTTGGGAGCGCCAGTTCCGTCTGTGTGTTCGAGTGGACAAAATGGCGAAGAT CGCCAAGACTCACGAAGGTAAGCGGTCTTTCCCTGCTTACG

CRISPR targeting EZH2 loci:

GGCTGGTTAGATTAGTGATTTTAATATGAAACCATTGCTTTTAGAATAAT

CATGGGCCAGACTGGGAAGAAATCTGAGAAGGGACCAGTTTGTTGGCGGA

CRISPR targeting PIK3CA loci:

CATCTAATTCCTTAAAGTAGTTTTATATGTAAAACTTGCAAAGAATCAGA

ACAATGCCTCCACGACCATCATCAGGTGAACTGTGGGGCATCCACTTGAT

GCCCCCAAGAATCCTAGTAGAATGTTTACTACCAAATGGAATGATAGTGA

CRISPR targeting SF3B1 loci:

GCGCAGAGTGCAGCCCCCAGCTATTTTTCTCCGTGGCGGCGGCGACGAGCGGAA GTTCTTGGGAGCGCCAGTTCCGTCTGTGTGTTCGAGTGGACAAAATGGCGAAGAT CGCCAAGACTCACGAAGGTAAGCGGTCTTTCCCTGCTTACG

Supplemental Methods:

Transfection and selection

Adherent cell lines: In a six well transfection reaction, 2ug of donor vector and 1ug of TALEN or CRISPR were co‐transfected using lipofectamine LTX (Lifetech). Shield‐1 (1uM, Clontech) was added the day after transfection. Three days post‐transfection, appropriate numbers of cells (to ensure single cell colonies after selection) were re‐seeded into 15cm plates, followed by puromycin selection (0.5ug/ml).

Fresh medium containing puro and Shld were changed every 4‐5 days. Single cell colonies were obtained from the 15cm plates after two or three weeks of puro selection and subsequently cultured and expanded in 96 well plates.

Suspension cell line (Karpas422): Transfection was conducted by NEON electroporation system

(LifeTech). Cells were maintained in culture medium containing Puro (0.5ug/ml) and Shield‐1 (1uM) after transfection for around 10 days, followed by single cell sorting into 96 well plates. Fresh medium containing Shield‐1 was added into the culture every 6 days.

Identification of positive Degron‐KI clones

Expanded clones were cultured in 24 well plates in the presence of 1uM Shld and collected for western blotting to identify positive targeting clones. The DD tag increases the molecular weight of the target protein by 12kd.

Genomic DNA isolation, RNA isolation, PCR, RT‐PCR, Quantitative Real‐Time PCR

Genomic DNA was purified using DNeasy Blood & Tissue Kit (Qiagen). RNA was purified using RNeasy plus mini kit (Qiagen). Genomic DNA PCR was conducted using standard Taq (lifetech) or Phire Hot Start

II DNA Polymerase (Thermo Scientific). RT‐PCR was conducted using SuperScript® III One‐Step RT‐PCR

System with Platinum® Taq High Fidelity (Lifetech). The detailed sequences of the primers are provided in Supplemental Experimental Procedures. Quantitative Real‐Time PCR was performed in triplicates using gene‐specific primers (p21 # 4331182 LifeTech) and FastStart Universal Probe Master Mix (Rox)

(Roche Applied Science) on an ABI 7900HT series PCR machine. Expression levels were normalized to

Actin expression.

Quantification of Western Blot result

Image J was used to quantify the band intensity in Supplemental Figure 4. Briefly, the area intensity of

DD tagged protein band subtracted the background intensity (from the blank area of the blot) and then normalized to GAPDH intensity. For every DD tagged clone, the intensity in the presence of Shld is set to be 1.0 and compared to the intensity in the absence of Shld.

RNA‐seq

Total RNA was quantified using the Agilent RNA 6000 Nano Kit (catalog number 5067‐1511) on the

Agilent 2100 BioAnalyzer. One microgram of high‐purity total RNA (RNA Integrity Number 9.8 or greater) was used as input to the Illumina TruSeq RNA Sample Prep Kit – Sets A/B (48Rxn) (catalog number FC‐

122‐1001 or FC‐122‐1002).

The gel‐free protocol was employed for the TruSeq RNA Sample Prep Kit per manufacturer’s specifications, and performed on the Beckman Coulter Biomek FXp robotics platform. The standard RNA fragmentation profile was used as recommended by Illumina (94 degrees Celsius for 8 minutes).

The PCR amplified RNA‐seq library products were then quantified using the Advanced Analytical

Fragment Analyzer Standard Sensitivity NGS Fragment Analysis Kit (catalog number DNF‐479). The samples were diluted to 10 nanomolar in Qiagen Elution Buffer (Qiagen material number 1014609), denatured, and loaded at 2.75 picomolar on an Illumina HiSeq2500 in Rapid Run mode using TruSeq

Rapid PE Cluster Kit – HS (catalog number PE‐402‐4001) and TruSeq Rapid SBS Kit – HS (200 cycle) reagents (catalog number FC‐402‐4001). The RNA‐seq libraries were sequenced at 100 base‐pair paired‐end with a 7‐base‐pair index using the standard Illumina primers. The sequence intensity files were generated on the instrument using the

Illumina Real Time Analysis software. The intensity files were demultiplexed and fastq files created using the CASAVA 1.8.2 software suite.

Sequencing reads were aligned to the reference with a modified version of Tophat as previously described (www.ncbi.nlm.nih.gov/pubmed/24362935). Transcripts were then assembled using CLASS version 1.05 (www.ncbi.nlm.nih.gov/pubmed/23734605), with UCSC KnownGene as reference transcripts; wt‐SF3B1‐specific‐DD‐KI samples were sequenced subsequently and were omitted from this step. Assembled transcripts from all samples and KnownGene transcripts were combined with

Cuffmerge (www.ncbi.nlm.nih.gov/pubmed/20436464), and this merged set served as the reference transcripts in subsequent steps. Expression values were calculated using Cufflinks version 2.2.1

(www.ncbi.nlm.nih.gov/pubmed/20436464) and rescaled with the 75th percentile gene expression value in each sample set to 10. Cuffdiff version 2.2.1 (www.ncbi.nlm.nih.gov/pubmed/20436464) was used to detect differential gene expression, and rMATS version 3.0.8

(www.ncbi.nlm.nih.gov/pubmed/25480548) was used to detect differential splicing. For simplicity, we recorded rMATS results based on both exon and junction counts, although results were consistent when only junctions were considered (not shown).

Sequencing reads and gene expression values were deposited in the NCBI Gene Expression Omnibus database.