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. Author manuscript; available in PMC: 2014 Oct 2.
Published in final edited form as: Cold Spring Harb Protoc. 2011 Jan 1;2011(1):pdb.prot5559. doi: 10.1101/pdb.prot5559

Table 1.

Comprehensive testing of different parameters of nanoCAGE method.

Library
name		 Reverse
Transcriptase
(RT)		 PCR
extension
time1(sec)		 RT
Temp
(°C)		 RT
additive2 		RT
Time
(min)		 RT
cool
Temp
(°C)3 		Total
tag
count4 		Unique
Tag
Count5 		rRNA6
(%)		 Redundanc
y7 		Semi-
suppressive
PCR cycles8 		Semi-
suppressive
PCR conc9
(ng/µl)
J53-GA PrimeScript 120 50 Betaine 30 No 1572426 181750 23.23 8.65 25 16.34
J53-GA PrimeScript 120 50 S/T 30 NO 2322991 359674 29.35 6.46 24 15.49
J53-GA PrimeScript 120 50 water 30 No 723881 108291 27.09 6.68 25 12.47
J53-GA PrimeScript 120 60 Betaine 30 No 1098557 40599 5.13 27.06 32 13.08
J53-GA PrimeScript 120 60 S/T 30 No 1917298 83125 4.27 23.07 33 14.22
J53-GA PrimeScript 120 60 water 30 No 2150491 68244 5.74 31.51 33 11.73
J53-GA PrimeScript 30 50 Betaine 30 No 1130096 112900 19.64 10.01 28 9.25
J53-GA PrimeScript 30 50 S/T 30 No 2359863 202012 26.46 11.68 28 8.17
J53-GA PrimeScript 30 50 water 30 No 1631842 125415 24.89 13.01 30 8.85
J53-GA PrimeScript 30 60 Betaine 30 No 665230 48412 5.09 13.74 32 9.28
J53-GA PrimeScript 30 60 S/T 30 No 1407092 34991 4.93 40.21 37 7.64
J53-GA PrimeScript 30 60 water 30 No 607120 20445 8.39 29.7 37 9.35
J54-GA SuperScriptIII 120 50 Betaine 30 No 250499 103388 16.86 2.42 23 28.8
J54-GA SuperscriptIII 120 50 S/T 30 No 1224346 215729 9.77 5.68 23 26.01
J54-GA SuperscriptIII 120 50 water 30 No 955875 300447 20.45 3.18 24 33.99
J54-GA SuperscriptIII 120 60 Betaine 30 No 927392 119713 12.76 7.75 26 20.3
J54-GA SuperscriptIII 120 60 S/T 30 No 205397 43212 16.31 4.75 27 20.42
J54-GA SuperscriptIII 120 60 water 30 No 1159443 109583 10.19 10.58 27 17.89
J54-GA SuperscriptIII 30 50 Betaine 30 No 574609 158085 15.67 3.63 28 32.93
J54-GA SuperscriptIII 30 50 S/T 30 No 859521 244124 11.17 3.52 27 14.27
J54-GA SuperscriptIII 30 50 water 30 No 508443 155528 22.29 3.27 28 13.11
J54-GA SuperScriptIII 30 60 Betaine 30 No 924509 68005 3.12 13.59 34 13.46
J54-GA SuperScriptIII 30 60 S/T 30 No 881722 119491 6.39 7.38 34 16.9
J54-GA SuperScriptIII 30 60 water 30 No 1842304 99336 3.12 18.55 33 21.13
J55-GA SuperScriptIII 120 50 Betaine 30 No 1210696 240266 14.36 5.04 25 26.78
J55-GA SuperScriptIII 120 60 S/T 30 No 2263452 443357 12.67 5.11 25 29.55
J56-GA SuperScriptIII 120 50 water 30 No 1678459 363513 20.77 4.62 24 22.81
J55-GA SuperScriptIII 120 60 Betaine 30 No 1617617 126028 5.52 12.84 30 16.12
J55-GA SuperScriptIII 120 60 S/T 30 No 549364 42617 3.28 12.89 30 14.5
J55-GA SuperScriptIII 120 60 water 30 No 431085 33658 7.8 12.81 30 14.96
J55-GA SuperScriptIII 30 50 Betaine 30 No 768911 182116 14.81 4.22 28 13.69
J55-GA SuperScriptIII 30 50 S/T 30 No 811168 238256 18.97 3.4 28 15.01
J55-GA SuperScriptIII 30 50 water 30 No 2034725 324021 18.14 6.28 30 15.84
J55-GA SuperScriptIII 30 60 Betaine 30 No 1474825 87977 3.38 16.76 36 10.76
J55-GA SuperScriptIII 30 60 S/T 30 No 783407 47124 4.63 16.62 36 12.53
J55-GA SuperScriptIII 30 60 water 30 No 1066288 57003 4.34 18.71 36 8.27
J61-GA SuperScriptIII 120 40 Betaine 30 No 1509574 489529 21.48 3.08 25 51
J61-GA SuperScriptIII 120 40 S/T 30 No 1241620 311979 23.19 3.98 25 29.08
J61-GA SuperScriptIII 120 40 water 30 No 1660969 499814 26.79 3.32 25 58.91
J61-GA SuperScriptIII 120 45 Betaine 30 No 2314533 588948 31.62 3.93 25 36.68
J61-GA SuperScriptIII 120 45 S/T 30 No 1194233 340681 22.45 3.51 25 36.03
J61-GA SuperScriptIII 120 45 water 30 No 1366263 332132 17.32 4.11 25 28.62
J61-GA SuperScriptIII 120 55 Betaine 30 No 1282996 181602 24.66 7.06 27 27.52
J61-GA SuperScriptIII 120 55 S/T 30 No 896055 227808 23.12 3.93 24 17.3
J61-GA SuperScriptIII 120 55 water 30 No 1336866 262873 22.77 5.09 27 28.33
J61-GA SuperScriptIII 30 40 Betaine 30 No 940078 301506 22.53 3.12 28 22.36
J61-GA SuperScriptIII 30 40 S/T 30 No 1294643 443824 21.7 2.92 28 27.6
J61-GA SuperScriptIII 30 40 water 30 No 1843239 473334 27.17 3.89 28 32.47
J61-GA SuperScriptIII 30 45 Betaine 30 No 743362 191280 22.1 3.89 30 18.71
J61-GA SuperScriptIII 30 45 S/T 30 No 951527 257689 22.14 3.69 30 22.99
J61-GA SuperScriptIII 30 45 water 30 No 695530 226251 16.97 3.07 30 27.02
J61-GA SuperScriptIII 30 55 Betaine 30 No 973648 159747 30.95 6.09 32 17.87
J61-GA SuperScriptIII 30 55 S/T 30 No 1201971 208873 32 5.75 30 19.96
J61-GA SuperScriptIII 30 55 water 30 No 1410042 165453 32.18 8.52 30 17.52
J62-GA SuperScriptIII 120 40 Betaine 30 No 94717 67342 15.42 1.41 28 51.91
J62-GA SuperScriptIII 120 40 S/T 30 No 143954 91610 8.71 1.57 26 75.86
J62-GA SuperScriptIII 120 40 water 30 No 177528 117916 15.15 1.51 25 42.51
J62-GA SuperScriptIII 120 45 Betaine 30 No 190529 99655 12.64 1.91 25 37.96
J62-GA SuperScriptIII 120 45 S/T 30 No 48803 30511 3.6 1.6 22 31.54
J62-GA SuperScriptIII 120 45 water 30 No 188310 121086 23.04 1.56 25 53.77
J62-GA SuperScriptIII 120 55 Betaine 30 No 157084 72413 2.71 2.17 27 11.16
J62-GA SuperScriptIII 120 55 S/T 30 No 15840 10994 2.5 1.44 27 16.92
J62-GA SuperScriptIII 120 55 water 30 No 78471 47392 1.38 1.66 27 15.22
J62-GA SuperScriptIII 30 40 Betaine 30 No 273149 188191 10.14 1.45 25 23.89
J62-GA SuperScriptIII 30 40 S/T 30 No 249603 186982 9.45 1.33 25 18.95
J62-GA SuperScriptIII 30 40 water 30 No 318088 245244 13.19 1.3 25 28.14
J62-GA SuperScriptIII 30 45 Betaine 30 No 124887 74796 7.21 1.67 25 16.8
J62-GA SuperScriptIII 30 45 S/T 30 No 94417 69055 13.34 1.37 25 19.86
J62-GA SuperScriptIII 30 45 water 30 No 34632 24388 3.91 1.42 25 18.91
J62-GA SuperScriptIII 30 55 Betaine 30 No 395583 132449 5.75 2.99 34 16.49
J62-GA SuperScriptIII 30 55 S/T 30 No 137211 65220 16.68 2.1 30 17.46
J62-GA SuperScriptIII 30 55 water 30 No 96976 25855 3.48 3.75 30 11.43
J63-GA PrimeScript 120 40 Betaine 30 No 1120560 516165 8 2.17 22 27.87
J63-GA PrimeScript 120 40 S/T 30 No 929189 442893 11.74 2.1 22 25.11
J63-GA PrimeScript 120 40 water 30 No 1305601 601415 10.01 2.17 22 28.33
J63-GA PrimeScript 120 45 Betaine 30 No 1368533 580968 17.64 2.36 22 18.73
J63-GA PrimeScript 120 45 S/T 30 No 649456 318800 15.27 2.04 22 20.76
J63-GA PrimeScript 120 45 water 30 No 739001 326532 18.53 2.26 22 19.22
J63-GA PrimeScript 120 55 Betaine 30 No 1003632 173383 22.31 5.79 29 25.95
J63-GA PrimeScript 120 55 S/T 30 No 1217507 303138 17.41 4.02 30 42.3
J63-GA PrimeScript 120 55 water 30 No 1987403 401085 17.62 4.96 30 25.45
J63-GA PrimeScript 30 40 Betaine 30 No 873112 428106 16.26 2.04 27 22.09
J63-GA PrimeScript 30 40 S/T 30 No 930354 443523 17.4 2.1 27 20.09
J63-GA PrimeScript 30 40 water 30 No 848335 426379 18.12 1.99 27 30.32
J63-GA PrimeScript 30 45 Betaine 30 No 574837 269437 21.96 2.13 28 26.98
J63-GA PrimeScript 30 45 S/T 30 No 881363 370124 20.19 2.38 28 23.45
J63-GA PrimeScript 30 45 water 30 No 312482 151889 13.84 2.06 28 17.7
J63-GA PrimeScript 30 55 Betaine 30 No 1170345 200170 12.99 5.85 38 42.86
J63-GA PrimeScript 30 55 S/T 30 N0 586387 127215 34.81 4.61 30 14.94
J63-GA PrimeScript 30 55 water 30 No 965347 119385 9.25 8.09 40 37.31
J64-GA SuperScriptIII 120 50 Betaine 30 30 722783 263787 28.9 2.74 25 55.85
J64-GA SSIII 120 50 Betaine 30 25 834995 286939 24.78 2.91 25 45.05
J64-GA SSIII 120 50 Betaine 30 20 1396843 319729 43.93 3.57 25 46.16
J64-GA SSIII 120 50 Betaine 15 No 1400473 347451 37.41 4.03 27 51.23
J64-GA SSIII 120 50 Betaine 45 No 110261 320173 48.44 3.44 24
J64-GA SSIII 120 50 Betaine 60 No 776672 266856 39.35 2.91 24
J64-GA SSIII 120 50 Betaine 30 30 917523 355315 26.38 2.58 25
J64-GA SSIII 120 50 Betaine 30 25 908191 371519 33.06 2.44 25
J64-GA SSIII 120 50 Betaine 30 20 787615 320633 27.46 2.46 25
J64-GA SSIII 120 50 Betaine 15 No 987514 348994 22.04 2.83 25
J64-GA SSIII 120 50 Betaine 45 N0 1387713 484912 33.68 2.86 25
J64-GA SSIII 120 50 Betaine 60 No 857554 331455 39.52 2.57 21
J64-GA PrimeScript 120 50 Betaine 30 30 1017950 274698 34.21 3.71 28
J64-GA PrimeScript 120 50 Betaine 30 25 907559 219400 47.38 4.14 27
J64-GA PrimeScript 120 50 Betaine 30 20 705686 190585 31.01 3.7 28
J64-GA PrimeScript 120 50 Betaine 15 No 1285912 270461 42.59 4.75 28
J64-GA PrimeScript 120 50 Betaine 45 N0 1143364 259958 54.18 4.4 23
J64-GA PrimeScript 120 50 Betaine 60 No 1191776 196585 27.24 6.06 26

Simple quality indicators for nanoCAGE libraries made with 50 ng of Hep G2 total RN, using different reverse-transcriptases and reaction conditions. that gave the best result with SuperScript III and PrimeScript. The libraries were sequenced on a Genome Analyzer II (Illummina).

1

Extension time of the semi suppressive PCR and the PCR to adapt linker to Illumina. Short incubation times were tested to reduce the size of the amplified cDNAs, since the Illumina GA II does not efficiently sequence cDNAs longer 1 kb in mixtures.

2

RT additive: S/T, Sorbitol/Trehalose; water, Nuclease-free water.

3

After RT reaction, the cDNAs were incubated for 5 min at the indicated temperatures before final cooling on ice/water mixture (see step 2 in the Method section). No: the cool down treatment was not applied to these combinations.

4

Number of tags remaining after removing the artifacts, primer-dimer etc. from the raw sequences.

5

Number of unique tags remaining after removing the artifacts, primer-dimer etc. from the raw sequences.

6

Percentage of total tags mapped to ribosomal RNA.

7

Total number of tags divided by the number of unique tags.

8

Number of PCR cycles required to amplify the purified cDNAs by semi-suppressive PCR (see step 4 in the Method section).

9

Concentration of the semi-suppressive PCR products after purification (see step 11 in the Method section).