2‘,3’ cyclic phosphate substrate no sub

strate

1 2 3 4 5

Supplemental figure 1

Reverse transcription oligonucleotide (adaptor complementarity)

PCR oligonucleotide (transcript complementarity)

0 20 40 60 80

005

001051

002052 Ala

AGC (n=353)CGC (n=85)TGC (n=64)

0 20 40 60 80

0004

0080021

ArgACG (n=1403)CCG (n=632)CCT (n=170)TCG (n=503)TCT (n=44)

0 20 40 60 80

0001

002003

004

AsnGTT (n=528)

0 20 40 60 80

00001

000200 03

00 04 AspGTC (n=4855)

0 20 40 60 80

001

0203

0405 Cys

ACA (n=1)GCA (n=144)

0 20 40 60 80

002

0406

08001

GlnCTG (n=221)TTG (n=279)

0 20 40 60 80

0005

00010002

GluCTC (n=6794)TTC (n=5919)

0 20 40 60 80

0001

003005

GlyCCC (n=2512)GCC (n=1218)TCC (n=427)

0 20 40 60 80

0002

004006

HisGTG (n=2050)

0 20 40 60 80

002

0406

0800 1 Ile

AAT (n=111)TAT (n=0)

0 20 40 60 80

005

001002

LeuAAG (n=68)CAA (n=126)CAG (n=571)TAA (n=41)TAG (n=29)

0 20 40 60 80

0002

0060001

LysCTT (n=2346)TTT (n=946)

0 20 40 60 80

005

001051

MetCAT (n=720)

0 20 40 60 80

002

0406

08021 Phe

GAA (n=268)

0 20 40 60 80

005

001051

ProAGG (n=31)CGG (n=211)TGG (n=361)

0 20 40 60 80

005

051052

0 53 SeC(e)

tRNA position

TCA (n=635)

0 20 40 60 80

02

46

801 Ser

AGA (n=5)CGA (n=7)GCT (n=33)TGA (n=5)

0 20 40 60 80

005

001051

00 2

Thr

tRNA position

AGT (n=195)CGT (n=348)TGT (n=33)

0 20 40 60 80

002

0406

0800 1 Trp

tRNA position

CCA (n=197)

0 20 40 60 80

001

0203

04 TyrGTA (n=45)

0 20 40 60 80

0002

004006

00 8 ValAAC (n=424)CAC (n=1764)TAC (n=598)

sdaer gnicn euqeS

tRNA position tRNA position

Supplemental figure 2

sdaer gn icneuqeS

sdaer gnic ne uqeS

sd aer g nic neu qeS

sdaer gnicne uqeS

1. TPT1 + NAD2. RNA fragmentation

+

p5’-

5’-p

5’-App

p5’- p

5’- p

ddC- 3’

ddC- 3’

ddC- 3’

ddC- 3’

+

+ ATP

PO4

+ Appr>p

1. tRNA Ligase2. Gel purification

Reverse Transcriptase

5’- p ddC- 3’- 5’3’-

RNA hydrolysis

5’- - 3’ 5’-App ddC- 3’+

5’- ddC- 3’

1. T4 RNA Ligase2. Gel purification

PCR and High-throughput Sequencing

Supplemental figure 3