Figure S2. The distribution of cofold structures in the sequenced random library and computer ... The RNA sequences were cofolded with the SR1 adapter.
SUPPLEMENTARY DATA Supplementary Data are available at NAR Online: Supplementary figures 1-2, Supplementary tables 1-5. Figure S1. Relative entropy at each position in the random region of sequenced libraries as plotted by enoLOGO. The y-axis represents the relative entropy in bits and the contribution from each nucleotide is proportional to the size of its letter. The x-axis represents the position of nucleotides in the random region from 5’ to 3’. “Stem loop 6” and “Stem loop 10” represent the random library data obtained from stem-loop based RT methods using 6 or 10 degenerate nucleotides in the RT primer respectively. Figure S2. The distribution of cofold structures in the sequenced random library and computer simulated randomized libraries. The RNA sequences were cofolded with the SR1 adapter sequence using Vienna RNAcofold. The 42-mer libraries contain the 21 nt constant sequence that is present in the RNA oligos at the 5’-end, while the 21-mer libraries lack the constant region. The distribution percentage of simulated 21-mer and 42-mer libraries was generated from an average of 10 and 3 simulated libraries, respectively. Each simulated library contains 300,000 computer generated random sequences. The numbers on the x-axis correspond to the cofold structures defined in Figure 5A and their generalized illustrations are shown underneath. Table S1. Oligo and adapter sequences. Table S2. miRNA sequences and their predicted secondary structures by CONTRAfold. Table S3. Sequencing information of Ion Torrent libraries. Table S4. Nucleotide frequencies at each position in random library and ligated libraries. PO represents the corresponding nucleotide position in the random region from 5’ to 3’. 1
Table S5. Predicted cofold structures of miRNA with SR1 or SR1-S adapter.
2
Table S1 Name Random RNA oligo A Random RNA oligo G Random RNA oligo C Random RNA oligo U Ligated RT Random A RT Random C RT Random G RT Random U RT SR1 SR1-S SR1-R
Sequence 5' to 3' GGGAGAGGGAUCACACAAUACNNNNNNNNNNNNNNNNNNNNA GGGAGAGGGAUCACACAAUACNNNNNNNNNNNNNNNNNNNNG GGGAGAGGGAUCACACAAUACNNNNNNNNNNNNNNNNNNNNC GGGAGAGGGAUCACACAAUACNNNNNNNNNNNNNNNNNNNNU CCTCTCTATGGGCAGTCGGTGATCAAGCAGAAGACGGCATACGA CCTCTCTATGGGCAGTCGGTGATGGGGGGGGGGGT CCTCTCTATGGGCAGTCGGTGATTTTTTTTTTTTTTTTTTTTTTTTTG CCTCTCTATGGGCAGTCGGTGATTTTTTTTTTTTTTTTTTTTTTTTTC CCTCTCTATGGGCAGTCGGTGATTTTTTTTTTTTTTTTTTTTTTTTTA rAPPTCGTATGCCGTCTTCTGCTTG-NH2 rAPPTCGTATGCCGTCACGGCTCGA-NH2 rAPPNNNNNNCCGTCTTCTGCTTG-NH2
IT Forward IT Reverse Stem Loop RT oligo A Stem Loop RT oligo C Stem Loop RT oligo G Stem Loop RT oligo T Stem Loop RT oligo A ext Stem Loop RT oligo C ext Stem Loop RT oligo G ext Stem Loop RT oligo T ext miR-103b New adapter miR-653 New adapter miR-567 New adapter miR-4803 New adapter miR-5183 New adapter miR-495 New adatper miR-712 New adatper
CCATCTCATCCCTGCGTGTCTCCGACTCAGGGGAGAGGGATCACACAATAC CCTCTCTATGGGCAGTCGGTGAT TCGTATGCCGTCTTCTGCTTGTACAGTAGCATACGANNNNNA TCGTATGCCGTCTTCTGCTTGTACAGTAGCATACGANNNNNC TCGTATGCCGTCTTCTGCTTGTACAGTAGCATACGANNNNNG TCGTATGCCGTCTTCTGCTTGTACAGTAGCATACGANNNNNT TCGTATGCCGTCTTCTGCTTGTACAGTAGCATACGANNNNNNNNNA TCGTATGCCGTCTTCTGCTTGTACAGTAGCATACGANNNNNNNNNC TCGTATGCCGTCTTCTGCTTGTACAGTAGCATACGANNNNNNNNNG TCGTATGCCGTCTTCTGCTTGTACAGTAGCATACGANNNNNNNNNT rAPPCATCCAAATGTCTTCTGCTTG-NH2 rAPPAACAGATTGTAGGCACCATCA-NH2 rAPPTTCTGTAGGCACCATCAATGA-NH2 rAPPCAGCCAAATGTCTTCTGCTTG-NH2 rAPPGACTCGCCGTCTTCTGCTTGA-NH2 rAPPGTACCCATCCAAATGTCTTCT-NH2 rAPPGTACCCATCCAAATGTCTTCT-NH2
Table S2. Name cel-let-7 dme-miR-11-3p hsa-miR-27a hsa-miR-31 pma-miR-103b rno-miR-122 mmu-miR-139-3p cel-miR-253 hsa-miR-519e hsa-mir-567 hsa-miR-611 bta-miR-653 gga-miR-1614 bta-miR-2419 hsa-miR-4803 bdi-miR 5183 mmu-mir-127 mmu-mir-495 mmu-mir-712 mmu-miR-101a mmu-miR-452-5p bmo-miR-2777 mdv1-miR-M10-3p osa-miR 1868 bmo-miR-3225 mml miR 30a-5p rno miR 151*
Sequence 5' to 3' CONTRAfold structure ...................... UGAGGUAGUAGGUUGUAUAGUU ..................... CAUCACAGUCUGAGUUCUUGC ......((((.......)))) UUCACAGUGGCUAAGUUCCGC ..((...((((())))).)). AGGCAAGAUGCUGGCAUAGCU ....((..........))..... AGCAGCAUUGUACAGGGCUAUGA ...................... UGGAGUGUGACAAUGGUGUUUG ...................... UGGAGACGCGGCCCUGUUGGAG ...................... UUAGUAGGCGUUGUGGGAAGGG ......(((......))).... AAGUGCCUCCUUUUAGAGUGUU .....(((((........))))) AGUAUGUUCUUCCAGGACAGAAC ....(((((((..)))))))... GCGAGGACCCCUCGGGGUCUGAC ..................... GUGUUGAAACAAUCUCUGUUG ......((((.......)))) GGCAUGGCAGACUCACCCUGC ...................... AUCGCAUCAACACUCGUCUGUU ...(((....)))........ UAACAUAAUAGUGUGGAUUGA ......(((........))). UAUUUGGACAAAUUUGAGUCA (((((....)))))........ UCGGAUCCGUCUGAGCUUGGCU ...................... AAACAAACAUGGUGCACUUCUU ...(........)........ CUCCUUCACCCGGGCGGUACC (((((())))))......... UACAGUACUGUGAUAACUGAA ......(((......))).... UGUUUGCAGAGGAAACUGAGAC ...(((((.....))))).. AGGGACCUUGCCUGGGUUAU .....(((((...))))).... UCGAAAUCUCUACGAGAUAACA ........................ UCACGGAAAACGAGGGAGCAGCCA ....................... UACAUAUGACUGUUUAUAACACU ........(((......))).. UGUAAACAUCCUCGACUGGAAG ..................... CUAGACUGAGGCUCCUUGAGG
Table S3.
Number of Reads Too short Keypass failure Mixed / Polyclonal Low Signal Poor Signal Profile Final Library Reads Reads after trimming Retained after collapsing
Number of Reads Too short Keypass failure Mixed / Polyclonal Low Signal Poor Signal Profile Final Library Reads Reads after trimming Retained after collapsing
Random oligos with 3'A: Poly C tailed Count % 708,506 --13,145 1.9% 31,895 4.5% 9,170 1.3% 5,293 0.75% 113,919 16% 534,919 75% 68,387 9.7% 68,310 99.9%
Random oligos with 3'G: Poly A tailed Count % 4,920,976 --74,356 1.5% 261,746 5.3% 64,708 1.3% 35,472 0.72% 769,167 16% 3,713,135 75% 638,688 13% 634,451 99.3%
Random oligos with 3'C: Poly A tailed Count % 838,297 --12,671 1.5% 62,682 7.5% 10,564 1.3% 28,745 3.43% 193,195 23% 529,961 63% 29,737 3.5% 29,211 98.2%
Random oligos with 3'U: Poly A tailed Count % 671,555 --9,265 1.4% 29,937 4.5% 4,692 0.7% 15,670 2.3% 78,434 12% 533,321 79% 67,800 10% 66,996 98.8%
Rnl 1
Rnl2tr
Rnl2tr K227Q
Rnl2tr R55K K227Q
Count 1,105,790 8,263 54,420 51,815 15,188 338,395 637,255 169,303 168,737
% --0.7% 4.9% 4.7% 1.4% 31% 58% 15% 99.7%
Rnl2tr R55K K225R K227Q Number of Reads Too short Keypass failure Mixed / Polyclonal Low Signal Poor Signal Profile Final Library Reads Reads after trimming Retained after collapsing
Count 633,976 6 20,152 10,261 N/A 103,000 500,557 213,778 213,184
% --0.0% 3.2% 1.6% 16% 79% 34% 99.7%
Count 459,605 9 19,954 6,114 N/A 55,055 378,473 190,016 189,649
% --0.0% 4.3% 1.3% 12% 82% 41% 99.8%
Random stem loop 6 Count 535,970 29 19,456 8,266 N/A 58,323 449,896 156,223 155,762
% --0.01% 3.6% 1.5% 11% 84% 29% 99.7%
Count 787,252 18 41,374 32,032 N/A 201,860 511,968 168,219 167,935
% --0.0% 5.3% 4.1% 26% 65% 21% 99.8%
Random stem loop 10 Count 529,088 3,122 24,896 7,792 4,987 74,716 413,430 43,094 43,004
% --0.59% 4.7% 1.5% 0.94% 14% 78% 8.1% 99.8%
Count 382,698 30 13,649 21,614 N/A 95,392 252,013 75,233 75,042
% --0.0% 3.6% 5.6% 25% 66% 20% 99.7%
Table S4. Random PO A C 1 25% 19% 2 24% 19% 3 25% 19% 4 25% 20% 5 25% 19% 6 24% 19% 7 25% 19% 8 24% 19% 9 24% 19% 10 25% 19% 11 25% 19% 12 24% 20% 13 24% 20% 14 25% 20% 15 25% 20% 16 24% 19% 17 24% 19% 18 25% 20% 19 23% 19% 20 22% 22% 21 25% 25%
G 33% 33% 33% 33% 33% 33% 33% 32% 32% 32% 32% 32% 32% 31% 31% 31% 31% 30% 34% 29% 25%
U 24% 24% 23% 23% 24% 24% 24% 25% 24% 24% 24% 24% 24% 24% 25% 25% 25% 25% 23% 26% 25%
Rnl1 PO 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21
A 25% 24% 25% 25% 25% 25% 24% 24% 24% 24% 24% 24% 25% 25% 25% 25% 26% 26% 27% 26% 28%
C 18% 18% 18% 18% 17% 17% 17% 18% 18% 19% 19% 19% 19% 19% 19% 19% 19% 19% 17% 16% 25%
G 29% 31% 31% 31% 32% 32% 32% 32% 32% 31% 31% 31% 31% 30% 30% 30% 29% 28% 29% 31% 22%
U 27% 27% 26% 26% 26% 26% 26% 26% 26% 26% 26% 26% 26% 26% 26% 26% 27% 27% 27% 27% 25%
Rnl2tr PO A 1 25% 2 25% 3 25% 4 25% 5 25% 6 25% 7 26% 8 26% 9 25% 10 26% 11 26% 12 25% 13 26% 14 26% 15 26% 16 26% 17 26% 18 26% 19 27% 20 25% 21 26%
C 18% 18% 19% 18% 18% 18% 18% 18% 18% 18% 18% 18% 18% 18% 18% 18% 19% 19% 16% 17% 27%
G 34% 34% 33% 33% 33% 33% 33% 32% 32% 32% 32% 32% 32% 31% 31% 31% 30% 30% 32% 32% 24%
U 23% 23% 23% 23% 24% 24% 24% 24% 24% 24% 24% 24% 25% 24% 25% 25% 25% 25% 24% 25% 23%
Rnl2tr K227Q PO A C 1 25% 18% 2 25% 18% 3 26% 18% 4 26% 18% 5 26% 18% 6 25% 18% 7 25% 18% 8 25% 18% 9 25% 18% 10 25% 18% 11 25% 19% 12 25% 19% 13 26% 19% 14 26% 19% 15 26% 19% 16 26% 19% 17 26% 19% 18 26% 19% 19 28% 16% 20 24% 17% 21 26% 29%
G 32% 32% 32% 32% 32% 32% 32% 32% 32% 32% 32% 31% 31% 31% 31% 30% 30% 28% 32% 32% 21%
U 25% 25% 24% 24% 24% 24% 24% 25% 25% 25% 25% 25% 25% 25% 25% 25% 26% 26% 25% 27% 25%
Rnl2tr R55K K227Q PO A C G 1 25% 18% 32% 2 25% 18% 32% 3 26% 18% 32% 4 26% 18% 32% 5 25% 18% 33% 6 25% 18% 33% 7 25% 18% 32% 8 25% 18% 32% 9 25% 18% 32% 10 25% 19% 31% 11 25% 19% 32% 12 25% 19% 32% 13 25% 19% 31% 14 25% 19% 31% 15 25% 19% 31% 16 26% 19% 30% 17 26% 19% 30% 18 26% 20% 28% 19 27% 16% 31% 20 24% 17% 32% 21 26% 27% 22%
U 25% 25% 25% 24% 24% 24% 25% 25% 25% 25% 25% 25% 25% 25% 25% 25% 26% 26% 25% 27% 25%
Rnl2tr R55K K225R K227Q PO A C G U 1 26% 18% 31% 25% 2 25% 19% 32% 24% 3 26% 18% 32% 24% 4 26% 18% 32% 24% 5 25% 18% 32% 25% 6 25% 18% 32% 25% 7 25% 18% 32% 25% 8 25% 18% 32% 25% 9 25% 19% 32% 25% 10 25% 19% 32% 25% 11 25% 19% 32% 25% 12 25% 19% 32% 25% 13 25% 19% 31% 25% 14 25% 19% 31% 25% 15 25% 19% 31% 25% 16 25% 19% 30% 26% 17 25% 19% 30% 26% 18 26% 19% 29% 26% 19 28% 16% 32% 24% 20 24% 17% 33% 26% 21 26% 29% 21% 24%
Table S5. miRNA/Adapter miR-31/SR1 miR-31/SR1-S miR-27a/SR1 miR-27a/SR1-S let7/SR1 let7/SR1-S miR-139-3p/SR1 miR-139-3p/SR1-S miR-122/SR1 miR-122/SR1-S miR-11-3p/SR1 miR-11-3p/SR1-S miR-253/SR1 miR-253/SR1-S miR-1614/SR1 miR-1614/SR1-S miR-2419/SR1 miR-2419/SR1-S miR-611/SR1 miR-611/SR1-S miR-103b/SR1 miR-103b/SR1-S miR-567/SR1 miR-567/SR1-S miR-5183/SR1 miR-5183/SR1-S miR-653/SR1 miR-653/SR1-S miR-495/SR1 miR-495/SR1-S miR-712/SR1 miR-712/SR1-S miR-452-5p/SR1 miR-452-5p/SR1-S miR-101/SR1 miR-101/SR1-S
Cofold structure (((((.((.((.((((((...&...))))))))..))))))). .(((..(.((.(((((((...&...))))))).))).)))... ....(((((((..........&......))))....))).... ......((((.......))))&(((...((((...)))).))) ..((((((.(((..(((((...&...)))))..))).)))))). ((((...((.((..(((((...&...)))))..))))..)))). .(((((((((((((....))..&..)).)).)))))))...... .(....).(((((.((.(((..&.......))).)).))).)). ..(((((.((..(((((((.((&.)).)))))))..))))))). ..(((((((((...(((((.((&.)).)))))))))).)))).. .....(((...((((...(((&..))).))..))..))).... ..(((.....)))........&(((...((((...)))).))) .(((.(((((..(((.((....&)).)))..))))).))).... ......((.(((((((...((.&.......)).))))))))).. ((((((((((.......))))&...))))))............ ((((((((((.......))))&...))))))............ ...(((((((((......))))&..).))))............. ........((((......))))&(((...((((...)))).))) ((((((((((....))))))...&))))..((.(....).))... ((((((((((....))))))...&))))..((((...)))).... (((((....(.((..((((((((&))))).))))))..))))).. .(.(((..((.((..((((((((&))))).)))))))..)))).. ............((((.(((((.&............))))))))) ....(((((.....)))))....&(((...((((...)))).))) .....((((..((.(((....&))).))...))))........ ......(((........))).&(((...((((...)))).))) ..((.(((((...........&.........)).))).))... .......((((.....)))).&(((...((((...)))).))) ...((((.(((((..((.....&..))..))))).)).)).... ...........((((.......&..))))((((...)))).... ...........((((((((..&.....))))))))........ .....((..((((((((((..&.....))))))).)))...)) .....((((((((..(...((.&))....)...))))))))... .((((.(((......)))))))&(((...((((...)))).))) ..(((.((.((.(((...((.&)).))))).))...))).... ..((((.........))))..&(((...((((...)))).)))
Structure # 5 5 5 2 5 5 7 5 9 9 9 14 13 5 11 11 11 2 15 15 1 1 5 14 13 14 5 14 5 5 5 5 13 2 13 14
