miR-98-5p

miR-28-3p
miR-23b-3p
miR-191-5p
Lys CTT tiR5
15

10
Log2
RPM

5
miR-148b-3p
Gln CTG tRF 3CC A
15

10

tRF3CCA
miR-194-1-5p

leader
miR-22-3p
18

16

14

12

10

tRF5 tRF1

tRF3

tRF5
iMet misc
8

4
Met tiR3

tiR5
tRF3CCA
Val CAC tiR5
L1/L2
leader
miR-92a-1-3p
S96

tRF1

tRF3
misc
S95

SeC
tiR3

tiR5

Phe
Asn
Asp
Cys

Leu
Arg

Gln
Glu

Pro

Ser
Lys
Gly

Thr
miR-199a-2-3p
Ala S94

His

Trp

Val
Tyr
Ile
S93
miR-423-3p S92
S96S96 S91
miR-30e-3p S95S95 S90
S94S94 S89
S93S93 S88
Gly GCC tRF5 S92S92 S87
S91S91 S86
let-7f-2-5p S90S90 S85
S89S89 S84
S88S88 S83
miR-125a-5p S87S87 S82
S86S86 S81
miR-101-2-3p S85S85 S80
S84S84 S79
S83S83 S78
miR-25-3p S82S82 S77
S81S81 S76
Top 50 expressed small RNAs

let-7c-5p S80S80 S75

S79S79 S74
S78S78 S73
miR-26b-5p
S77S77 S72
S76S76 S71
Ser TGA tRF1 S75S75 S70
S74S74 S69
S73S73 S68
miR-30a-5p
S72S72 S67
S71S71 S66
let-7b-5p S70S70 S65
S69S69 S64
S68S68 S63
miR-151a-3p
S67S67 S62
S66S66 S61
miR-92a-3p S65S65 S60
S64S64 S59
S63S63 S58
Gly CCC tiR5
S62S62 S57
S61S61 S56
miR-26a-2-5p S60S60 S55
S59S59 S54
S58S58 S53
let-7a-2-5p
S57S57 S52
S56S56 S51
miR-378a-3p S55S55 S50
S54S54 S49
S53S53 S48
S47
miR-100-5p
S52S52 S46
miR-10a-5p S51S51 S45
S50S50 S44
S49S49 S43
let-7i-5p S48S48 S42
S47S47 S41
let-7a-5p S46S46 S40
S45S45 S39
S44S44 S38
let-7f-1-5p S43S43 S37
S42S42 S36
miR-146b-5p S41S41 S35
S40S40 S34
S39S39 S33
miR-30d-5p S38S38 S32
S37S37 S31
miR-27b-3p S36S36 S30
S35S35 S29
S34S34 S28
miR-126-3p S33S33 S27
S32S32 S26
Supplementary Figure 1

let-7g-5p S31S31 S25

S30S30 S24
S29S29 S23
S28S28
let-7f-5p
S27S27 S22
S26S26 S21
miR-26a-5p
S25S25 S20
S24S24 S19
miR-99a-5p
S23S23 S18
S22S22 S17
S21S21 S16
miR-143-3p
S20S20 S15
S19S19 S14
Gly GCC tiR5
S18S18 S13
S17S17 S12
S16S16 S11
miR-21-5p
S15S15 S10
S9
Glu CTC tiR5 S14S14 S8
S13S13 S7
S12S12 S6
S5
miR-192-5p S11S11 S4
S10S10 S3
miR-148a-3p S9 S9 S2
S8 S8 S1
S7 S7
miR-122-5p S6 S6
S5 S5
S4 S4
S3 S3

100000
S2 S2

10
10000

1
10000000

1000
1000000

100
S1 S1

B
A
Norm. count
Supplementary Figure 1. Abundance of tRFs in donor liver transplant biopsy

samples.

(A) Bar plot displaying the expression (DESeq2 normalized counts) of the top fifty most

highly expressed small RNAs in donor liver biopsy samples. These top fifty genes consist

entirely of miRNAs (gray bars) and tRFs (red bars). (B) Heatmap showing tRF expression

across liver biopsy samples (L1 and L2), organized by the amino acid carried by the

parental tRNA. Expression values are displayed as log2 RPM. tRFs derived from Glu-

and Gly-carrying tRNAs are highly enriched relative to other amino acid isotypes.
Supplementary Figure 2
A
Group EAD NF p value

Mean INR (Day 1 post transplant) 2.10 2.03 N.S.

Mean AST (Day 7 post transplant) 130.3 70.7 N.S.

Mean ALT (Day 7 post transplant) 235.4 121.9 0.017 *

Mean INR (Day 7 post transplant) 1.22 1.16 N.S.

B Gly CCC tiR5 C Tyr GTA tRF1 D Arg ACG tRF1

0.025 10000 0.89 0.029
0.97 0.23 0.29
0.48 group 5e−11 group 0.00081 group
1e+06 1000
0.0092 EAD L1 9e−10 EAD L1 0.14 E

Norm. count (log10)

Norm. count (log10)

Norm. count (log10)

1000L2
EAD EAD L2 E
NF L1 NF L1 N
100
1e+05 NF L2 NF L2 N

100
Type Type Type
L1 10 L1
L1
1e+04
L2 L2 L2

10
1

EAD L1 EAD L2 NF L1 NF L2 EAD L1 EAD L2 NF L1 NF L2 EAD L1 EAD L2 NF L1 NF L2

group group group

E F
DOWN-regulated in L2 (post) vs L1 (pre) Gln CTG tRF3CCA
1e+05 0.81
0.23
EAD NF 1.5e−14 group
Val TAC tRF1
1.3e−09 E
Ser GCT tRF3
Norm. count (log10)

Ile TAT misc 7 30 0 E

Ile AAT tRF3CCA
N
miR-4633-5p
His GTG leader 1e+04 N
miR-4521-5p

Gln CTG tRF3CCA, Gln CTG tRF3, Gln TTG tRF3CCA, Gln TTG tRF3, Ser CGA Type
tRF3, Ser CGA tRF3CCA, miR-5100-3p, Leu CAA tRF3CCA, Pro AGG tRF3, Pro L
TGG tRF3, Leu CAA tRF3, Pro CGG tRF3, Thr TGT tRF3CCA,
L
Ser AGA RF3CCA, Trp CCA tRF3CCA, Ser TGA tRF3CCA, Pro AGG tRF3CCA,
Pro TGG tRF3CCA, Pro CGG tRF3CCA, Ser AGA tRF3, Tyr GTA tRF3CCA, Leu
CAG tRF3CCA, Thr TGT tRF3, Trp CCA tRF3, Ser TGA tRF3, Ser GCT
tRF3CCA, Thr CGT tRF3CCA, Leu AAG tRF5, Arg TCG tRF3CCA, Leu TAG tRF5 1e+03
EAD L1 EAD L2 NF L1 NF L2
group
Supplementary Figure 2. Additional outcome-specific IRI-associated tRF

alterations.

(A) Table of additional post-transplant clinical parameters, stratified by patient transplant

outcome (EAD or NF). (B—D, F) Boxplots showing the normalized expression (log10

transformed) of selected tRF species across outcome groups (EAD or NF) and biopsy

timepoints (L1 or L2). The selected tRF species represent examples of changes between

L2 and L1 samples that are specific to EAD, NF, or shared by both groups. (B) Gly-CCC-

tiR5 is significantly upregulated in L2 compared to L1 biopsies, exclusively within EAD

samples. (C) Try-GTA-tRF1 is significantly upregulated in L2 across both EAD and NF

samples. (D) Arg-ACG-tRF1 is significantly upregulated in L2 samples only within the NF

group. (E) Differential expression analysis of L2 vs. L1 samples was performed separately

within EAD and NF groups. The Venn diagram displays small RNAs downregulated in L2

relative to L1 for each group. In the EAD group, uniquely downregulated RNAs included

multiple tRF subtypes and miRNA species. NF samples exhibited no uniquely

downregulated RNAs, with all L2-downregulated species overlapping with those in the

EAD group, the majority being tRF3CCA species. (F) Gln-CTG-tRF3CCA is

downregulated in the L2 sample across both EAD and NF groups, exemplifying the

overlapping downregulated tRF3CCA species presented in panel E.

Supplementary Figure 3
A UP-regulated (EAD vs NF)
Ala TGC tRF3 L1 L2 SeC TCA misc miR-12136-5p
Ala CGC tRF3 miR-8069-2-3p miR-3178-5p
Ala AGC tRF3 Val AAC tiR5 miR-32-5p
miR-328-3p Glu TTC tiR3 miR-484
miR-331-3p Glu TTC tiR5 miR-629-5p
miR-1180-3p 23 10 32 miR-144-3p miR-4430-3p
miR-5010-3p miR-183-5p Val AAC misc
miR-145-5p miR-1843-5p miR-107-3p
miR-128-1-3p miR-500a-3p miR-210-3p
miR-502-3p Gly GCC misc miR-342-3p
miR-128-2-3p miR-592-5p Gly TCC tiR5
miR-92a-2-3p miR-452-5p Val CAC misc miR-885-3p
miR-29b-1-3p miR-224-5p Gly CCC tiR5 miR-369-3p
miR-95-3p miR-874-3p Gly CCC misc miR-136-3p
miR-24-1-3p miR-24-1-5p Asp GTC tiR3 miR-182-5p
miR-504-5p miR-664-3p miR-22-5p
let-7e-3p miR-34a-5p miR-142-3p
miR-128-3p miR-27b-3p
miR-330-3p miR-664a-3p
miR-1307-3p miR-1301-3p
miR-199a-2-5p miR-484-5p
miR-744-5p
miR-199a-2-3p

B
DOWN-regulated (EAD vs NF)
L1 L2
Pro AGG tiR5 miR-212-5p miR-424-3p His GTG tRF1
Pro TGG tiR5 His GTG tiR5 Arg TCG tRF1 Ser AGA tRF5
SeC TCA tiR5 Gly TCC tiR5 miR-320a-5p miR-92a-3p
Asp GTC tiR5 Asp GTC misc His GTG leader Ser TGA tRF5
Gly CCC tiR3 Gly TCC tRF5 42 5 39 Arg ACG tRF3CCA Asp GTC tRF1
Glu TTC tiR5 miR-30b-3p Cys GCA tRF1 Gly GCC tRF3
Lys CTT tiR5 miR-27a-5p Ile AAT tRF1 miR-6715b-3p
Val TAC tiR5 SeC TCA tRF5 iMet CAT misc Gly TCC tRF3
Lys TTT tiR5 miR-12136-5p Lys CTT tRF5 miR-1268a
Ser GCT tRF5 Tyr GTA tRF5 miR-4521-5p Val AAC tRF1
Glu TTC tRF5 Gly CCC tiR5 Gly TCC tRF3CCA miR-92a-2-3p miR-192-3p
miR-1304-3p let-7f-2-3p Pro CGG tiR5 Ser GCT tRF1 miR-382-5p
miR-8069-2-3p miR-548e-3p Trp CCA tRF3CCA Thr CGT tRF1 miR-675-5p
miR-1246-5p Arg CCG misc miR-378f-3p Trp CCA tRF3 miR-1303-3p
Gly GCC tRF5 Leu CAG tRF5 Gly GCC tRF3CCA Val TAC tRF1 miR-92a-1-3p
Val CAC tiR5 Cys GCA misc Arg ACG tRF1 Lys TTT tiR3
Gly CCC tRF5 miR-1295b-3p Tyr GTA misc Arg CCT tRF1
miR-378d-2-5p miR-92a-1-5p Val CAC tRF1 Ser CGA tRF3CCA
let-7a-1-5p His GTG tRF5 Ser TGA tRF1 miR-151b-3p
Gly GCC tiR5 Glu CTC tRF5 Pro TGG misc
Glu TTC tiR3
Val TAC tiR3
Supplementary Figure 3. Differential expression of small RNAs between EAD and

NF groups.

(A) and (B) Differential expression analysis was performed separately within the L1 and

L2 sample sets, comparing EAD vs. NF transplant outcomes. (A) Venn diagram showing

small RNAs upregulated in EAD compared to NF at either the L1 or L2 timepoint. Ten

species were commonly upregulated across both timepoints, all of which were miRNAs.

(B) Venn diagram showing small RNAs downregulated in EAD compared to NF at either

the L1 or L2 timepoint. Five species were commonly downregulated across both

timepoints, the majority of which were tRF3CCA fragments, along with one tiR5 and one

miRNA species.
Supplementary Figure 4
A Perfusate sample summary (n = 161)
Group EAD NF p value
Number of patients 49 112

Recipient mean age 58.7 58 N.S.

Recipient gender (F/M) 15/34 42/68 N.S.

Donor mean age 45.6 39.8 0.011 *

Donor gender (F/M) 14/35 55/57 0.024 *

Mean CIT (hours) 4.56 4.51 N.S.

Mean AST (Day 1 post transplant) 4644 826 2.683e-10 ***

Mean ALT (Day 1 post transplant) 1746 458 7.382e-11 ***

Mean INR (Day 1 post transplant) 1.87 1.83 N.S.

Mean AST (Day 7 post transplant) 86 86.5 N.S.

Mean ALT (Day 7 post transplant) 211 123 8.385e-06 ***

Mean INR (Day 7 post transplant) 1.20 1.18 N.S.

B tissue
perfusate
NEBNext sRNA

NEBNext sRNA
library marker

library marker
50bp marker

50bp marker
LUT044L1

LUT044L2

LUT071L1

LUT071L2

LUT085L1

LUT085L2

UTL145

UTL082

UTL124

UTL080

UTL085

UTL129

242
217 242
190 217
160 190
147 150 160 150
123 147
110 123
100 110 100
90
90
76 76
67 67

50
50
Supplementary Figure 4. Clinical cohort and cDNA library characteristics.

(A) Summary of demographic and clinical characteristics for the cohort from which

perfusate samples were obtained (n = 161), stratified by outcome group (EAD or NF) (B)

Representative PAGE gels showing small RNA cDNA library preparations from tissue

biopsies (left) and perfusate samples (right). Fragments of ~150 bp (highlighted by blue

box) were excised and used for small RNA sequencing.

Supplementary Figure 5
A
Pearson’s correlation between perfusate and tissue small RNAs
Patient: 0
LUT005 LUT007 LUT023 LUT024 LUT039 LUT010 LUT045 LUT072 LUT046 LUT004 LUT014 LUT015 LUT044 LUT085 LUT088 LUT099 LUT052 LUT057

Perfusate ~ L1 0.69 0.70 0.63 0.73 0.69 0.71 0.68 0.75 0.73 0.5
0.74 0.67 0.76 0.68 0.71 0.75 0.75 0.74 0.69
Perfusate ~ L2 0.73 0.70 0.71 0.74 0.76 0.74 0.75 0.75 0.79 0.76 0.68 0.64 0.70 0.73 0.76 0.77 0.77 0.75 1

Patient: 0
LUT061 LUT070 LUT073 LUT048 LUT051 LUT083 LUT087 LUT106 LUT065 LUT077 LUT084 LUT105 LUT089 LUT111 LUT117 LUT127 LUT038 LUT098

Perfusate ~ L1 0.85 0.72 0.70 0.66 0.67 0.63 0.59 0.68 0.75 0.5
0.65 0.76 0.62 0.56 0.69 0.74 0.76 0.73 0.68
Perfusate ~ L2 0.82 0.66 0.68 0.72 0.70 0.67 0.65 0.67 0.79 0.69 0.73 0.69 0.67 0.71 0.76 0.77 0.74 0.73 1

B LUT005 C
−5 0 5 10 15

UP-regulated (EAD vs NF)

L1 15
10
5
0

Perfusate 18 0 32 L2
−5
−10

0 10 miR-452-5p
15

miR-224-5p
*** miR-874-3p
10

R = 0.959 L2 23 miR-24-1-5p
5

miR-664-3p
miR-34a-5p
0

miR-27b-3p
−5

L1 miR-664a-3p
15

miR-1301-3p
*** *** miR-484-5p
10

R = 0.692 R = 0.733 Perfusate

5
0
−5

−10 −5 0 5 10 15
Log2 (RPM) −5 0 5 10 15

D
T
ge

T
AS

AL
ra

1
1
no

y
y
IT
do

da
da
C

Pro−CGG−tiR5 −0.09 0.08 −0.27 −0.31 0.8

0.6

Pro−TGG−tiR5 −0.09 0.06 −0.27 −0.30 0.4

0.2

Pro−AGG−tiR5 −0.08 0.07 −0.27 −0.32 0

−0.2

Ser−TGA−tRF1 0.01 0.10 −0.29 −0.21 −0.4

−0.6

Pro−TGG−misc −0.06 −0.02 −0.22 −0.25 −0.8

−1
Supplementary Figure 5. Donor liver perfusate and tissue small RNA profiles are

correlated and may be relevant to EAD.

(A) Pearson correlation coefficients between perfusate and paired donor liver biopsy

samples (L1 and L2). (B) Representative pairwise scatterplots of small RNA expression

(log2 RPM) in patient LUT005 demonstrate a strong correlation between L1 and L2 biopsy

samples (R = 0.959), and moderate correlations between perfusate and L1 (R = 0.692)

or L2 (R = 0.733) biopsies. (C) Venn diagram comparing small RNAs upregulated in EAD

versus NF across three sample types: perfusate, L1 biopsy, and L2 biopsy. No small

RNAs were common to all three, while ten miRNAs were consistently upregulated in EAD

at both L1 and L2 timepoints. (D) Heatmap showing correlation coefficients (Pearson’s R)

between selected perfusate tRF candidates and clinical variables. “X” symbols indicate

non-statistically significant correlation coefficients. Multiple tRFs were negatively

correlated with day 1 AST and ALT values, while no consistent associations were

observed with donor age or CIT.