Figure S5

Figure S1 A.

B. 1.

Proteinase K digest

G-term

Polyadenylation

RBP

3’

5’

3’

5’

A-term

3’

RBP

2.

U-term

Reaction time

AAAAAAAAA

5’

C-term

polyadenylated fraction

Reverse transcription AAAAAAAAA VTTTTTT Illumina adaptor

AAAAAAAAA VTTTTTT Illumina adaptor

input (21 mer RNA oligo)

ATP concentration 3.

A

B

G-tailing

Circular ligase

C

IVT (in vitro transcription) AAAAA T7-pol VTTTTTT

GGGGG

TTTTTTV Illumina adaptor

UUUU UUUU UUUU

J. Ule lab protocol (Huppertz et al. 2013)

PCR

Linearization and PCR

polyadenylated fraction

T7complement T7

Illumina adaptor


Adaptor ligation Reverse transcription and PCR

C.

D.

Antibodies used: SC-28201 SCBT - - - + - + + + - + -

C.

U-term U-term


A301-093A A301-487A A301-486A Bethyl Bethyl Bethyl - - - + - - - + - - - + - + + - + + - + + + - + + - + + - + -

anti-CSTF2/2tau anti-CSTF2

Western Blot siLuc (mock control) siCSTF2 siCSTF2tau

anti-CSTF2tau Ponceau stain (loading)

g g g 1μ 1μ 5μ

0,

E.

F.

ut

10

%

inp

-

+

-

+

-

+

+

-

+

-

+

-

IP Antibody CSTF2tau Beads only

+

+

-

-

-

-

-

-

-

-

+

+

SDS Empigen

-

-

+

+

-

-

NP-40

-

anti CSTF2tau

-

+

IgG

kDa 250 150 100 75

α-CSTF77 α-CSTF2tau

*

+

CSTF2tau

50 37

G.

H. +++

135 113

+++

+++

+

+

-

+

+

-

+

+

+

1

2

3

4

RNaseT1 AB UV

in vitro transcribed RNA

200 nt

PCR product ready for sequencing

300 bp 200 bp

CS

G Ig

TF 2

ta

u

55

I. 6

sCLIP R1 (CSTF2tau)

Usable (non-PCR duplicated) reads (x106)

R² = 1 R² = 0.99

5

sCLIP R2 (CSTF2tau) eCLIP R1 (IGF2BP1)

R² = 0.99

eCLIP R2 (IGF2BP1)

4

iCLIP R1 (IGF2BP1)

R² = 0.99

iCLIP R2 (IGF2BP1) 3

2

R² = 0.99

1

R² = 0.97

0 0

1

2

3

4

5 6

Uniquely mapped reads (x10 )

6

Figure S2

A.

B. 1 FASTQ Processing

2 Map to hg 19 (STAR)

Files for visualization with Integrated Genome Viewer

3

Adapter removal 4

Reads Quality assessment: FASTQC Alignment: STAR with replicates and different treatment

with replicates

no replicates

Collapsing duplicate reads Peak calling: CLIPper

5

Merge replicates Call clusters (CLIPper)

Merge replicates Call clusters (CLIPper)

Call clusters (CLIPper)

Merge Replicates CLIPper cluster file {Chr}{Start}{Stop}{Cluster_name}{min_pval}{strand}{thick_start}{thick_end}

Annotate peaks to genome features

Gene lists 6 Remove low p-val peaks

Motif analysis

non-coding RNA

7 Subtract background peaks 8 Annotate peaks to genomic regions

3’UTR

5’UTR

introns

exons

C. 9 Replicates consistency analysis

Sites distribution consistency plot

Motif Analysis 10 Nucleotide / Dinucleotide distribution analysis (HOMER)

Percentage

Nucleotide frequencies A frequency C frequency G frequency T frequency

bar diagram pie diagram

weblogo nucleotide frequency plot

11 Differential binding analysis list of differentially bound sites

sites plot

E. Motif sequence

Dinucleotide frequencies AA frequency AT frequency GT frequency TG frequency TT frequency

Percentage

D.

Distance from binding site (nt)

Distance from binding site (nt)

Number of sites in bound seq

Number of sites in all seq

p-val

e-val

TGTGTV

367

5165

1.4e-15

8.1e-12

GSCGCC

37

309

1.4e-6

8.0e-3

TGSTTG

134

1865

3.6e-6

2.0e-2

GGTGYC

86

1059

3.6e-6

2.0e-2

0.20

F.

G.

3'end CLIP reads frequency

snoRNA; 12 0.15

sense intronic; 24 0.10

sense overlapping; 5 pseudogene; 36

polymorphic pseudogene; Mt 1 Mt rRNA; 2 tRNA; 2 0.05

3 prime overlapping snRNA; 7 ncrna; 1 an sense; 63

lincRNA; 67 miscRNA; 1

0 −100

−50

0

50

100

Distance to cleavage site (nt)

H. Replication-dependent histones: HIST1H4E HIST1H3D

Replication-independent histones: H1FX 160 nt H3F3A 142 nt

CSTF2tau conCLIP Rep1






CSTF2tau conCLIP Rep4 RefSeq polyA site annotation

RefSeq

CDS

CDS

Figure S3 A.

B. CSTF2tauKD Rep1

1

CSTF2tau KD2 control 1

0.996 0.998 0.985

control 2

1 0.996 0.986

1

u 2ta

TF

CS

2

KD

u 2ta

TF

CS

KD

1 0.994

1

l1

l2

tro con

control Rep2

BCV distance 2

CSTF2tau KD1

tro con

control Rep1

CSTF2tauKD Rep2

BCV distance 1

C.

D. noncoding; 37

FDR0.05

fold change

protein coding; 168

an sense ; 4 lincRNA;

snRNA; 5 Average log2 CPM

8

E. Gene type Sn/snoRNAs Other Genes

Positively regulated Negatively Regulated 56 0 575 463

snoRNA; 15

miscRNA ;3 pseudogene;2

G.

F.

*

0,9 0,8

SNORA20

RNU11 CSTF2tau KD Rep1 CSTF2tau KD Rep2

50 0

700 0 700

520

0

0 50 0

700

350 50

0

0

RNU11

SNORA20

mock control Rep2

46,45

50

700 mock control Rep1 0

0,7 0,6 0,5 29,1

*

* * *

*

SNORA20 SNORA14B

0,4

SNORA79

0,3

SCARNA18 SNORD89

0,2 0,1

n=3

0 SNORA20 SNORA14B SNORA79 SCARNA18 SNORD89

Figure S4 ePAT and PCR

A. U5

U4

U11 U4ATAC OAZ1 (positive control)

U1

polyA amplicon

B.

+

+

+ -

+ -

+ -

+ -

+ -

siLuc (mock control) siRNA 1 CSTF2tau siRNA 2 CSTF2tau

Loading control (Ponceau)

0% 0% 5% 5 2

10 C.

ActD treatment GAPDH vs. ACTB

*

160 140

n.s.

n.s.

120 100 80

mock control

60

CSTF2tau deple on

40

n=5

20 0

0h

2h

4h

6h

Figure S5

exon skipping

ANK2

exon38

exon39

exon retention

RAP1GDS1

exon13

intron skipping

CNRIP1

intron

exon2

exon14/3‘UTR