Table

Table 1. Comprehensive testing of different nanoCAGE parameters

Library
name 		Reverse
transcriptase(RT) 		PCR
ext.time(sec) a		 RT
temp(°C) 		RT
additiveb		 RT
time(min) 		RT
cooltemp(°C)c		 Total
tagsd		 Unique
tagse		 rRNA
(%)f		 Redundancyg Semisuppr.
PCRcyclesh		 Semisuppr.
PCRconc.(ng/μL)i
J53-GA PrimeScript 120 50 betaine 30 --- 1,572,426 181,750 23.23 8.65 25 16.34
S/T 30 --- 2,322,991 359,674 29.35 6.46 24 15.49
water 30 --- 723,881 108,291 27.09 6.68 25 12.47
60 betaine 30 --- 1,098,557 40,599 5.13 27.06 32 13.08
S/T 30 --- 1,917,298 83,125 4.27 23.07 33 14.22
water 30 --- 2,150,491 68,244 5.74 31.51 33 11.73
30 50 betaine 30 --- 1,130,096 112,900 19.64 10.01 28 9.25
S/T 30 --- 2,359,863 202,012 26.46 11.68 28 8.17
water 30 --- 1,631,842 125,415 24.89 13.01 30 8.85
60 betaine 30 --- 665,230 48,412 5.09 13.74 32 9.28
S/T 30 --- 1,407,092 34,991 4.93 40.21 37 7.64
water 30 --- 607,120 20,445 8.39 29.7 37 9.35
J54-GA SuperScript III 120 50 betaine 30 --- 250,499 103,388 16.86 2.42 23 28.8
J54-GA SuperScript III 120 50 S/T 30 --- 1,224,346 215,729 9.77 5.68 23 26.01
water 30 --- 955,875 300,447 20.45 3.18 24 33.99
60 betaine 30 --- 927,392 119,713 12.76 7.75 26 20.3
S/T 30 --- 205,397 43,212 16.31 4.75 27 20.42
water 30 --- 1,159,443 109,583 10.19 10.58 27 17.89
30 50 betaine 30 --- 574,609 158,085 15.67 3.63 28 32.93
S/T 30 --- 859,521 244,124 11.17 3.52 27 14.27
water 30 --- 508,443 155,528 22.29 3.27 28 13.11
60 betaine 30 --- 924,509 68,005 3.12 13.59 34 13.46
S/T 30 --- 881,722 119,491 6.39 7.38 34 16.9
water 30 --- 1,842,304 99,336 3.12 18.55 33 21.13
J55-GA SuperScript II 120 50 betaine 30 --- 1,210,696 240,266 14.36 5.04 25 26.78
S/T 30 --- 2,263,452 443,357 12.67 5.11 25 29.55
water 30 --- 1,678,459 363,513 20.77 4.62 24 22.81
60 betaine 30 --- 1,617,617 126,028 5.52 12.84 30 16.12
S/T 30 --- 549,364 42,617 3.28 12.89 30 14.5
water 30 --- 431,085 33,658 7.8 12.81 30 14.96
30 50 betaine 30 --- 768,911 182,116 14.81 4.22 28 13.69
S/T 30 --- 811,168 238,256 18.97 3.4 28 15.01
water 30 --- 2,034,725 324,021 18.14 6.28 30 15.84
60 betaine 30 --- 1,474,825 87,977 3.38 16.76 36 10.76
S/T 30 --- 783,407 47,124 4.63 16.62 36 12.53
water 30 --- 1,066,288 57,003 4.34 18.71 36 8.27
J61-GA SuperScript III 120 40 betaine 30 --- 1,509,574 489,529 21.48 3.08 25 51
S/T 30 --- 1,241,620 311,979 23.19 3.98 25 29.08
water 30 --- 1,660,969 499,814 26.79 3.32 25 58.91
45 betaine 30 --- 2,314,533 588,948 31.62 3.93 25 36.68
S/T 30 --- 1,194,233 340,681 22.45 3.51 25 36.03
water 30 --- 1,366,263 332,132 17.32 4.11 25 28.62
55 betaine 30 --- 1,282,996 181,602 24.66 7.06 27 27.52
S/T 30 --- 896,055 227,808 23.12 3.93 24 17.3
water 30 --- 1,336,866 262,873 22.77 5.09 27 28.33
30 40 betaine 30 --- 940,078 301,506 22.53 3.12 28 22.36
S/T 30 --- 1,294,643 443,824 21.7 2.92 28 27.6
water 30 --- 1,843,239 473,334 27.17 3.89 28 32.47
45 betaine 30 --- 743,362 191,280 22.1 3.89 30 18.71
S/T 30 --- 951,527 257,689 22.14 3.69 30 22.99
water 30 --- 695,530 226,251 16.97 3.07 30 27.02
55 betaine 30 --- 973,648 159,747 30.95 6.09 32 17.87
S/T 30 --- 1,201.971 208,873 32 5.75 30 19.96
water 30 --- 1,410,042 165,453 32.18 8.52 30 17.52
J62-GA SuperScript II 120 40 betaine 30 --- 94,717 67,342 15.42 1.41 28 51.91
S/T 30 --- 143,954 91,610 8.71 1.57 26 75.86
water 30 --- 177,528 117,916 15.15 1.51 25 42.51
45 betaine 30 --- 190,529 99,655 12.64 1.91 25 37.96
S/T 30 --- 48,803 30,511 3.6 1.6 22 31.54
water 30 --- 188,310 121,086 23.04 1.56 25 53.77
55 betaine 30 --- 157,084 72,413 2.71 2.17 27 11.16
S/T 30 --- 15,840 10,994 2.5 1.44 27 16.92
water 30 --- 78,471 47,392 1.38 1.66 27 15.22
30 40 betaine 30 --- 273,149 188,191 10.14 1.45 25 23.89
S/T 30 --- 249,603 186,982 9.45 1.33 25 18.95
water 30 --- 318,088 245,244 13.19 1.3 25 28.14
45 betaine 30 --- 124,887 74,796 7.21 1.67 25 16.8
S/T 30 --- 94,417 69,055 13.34 1.37 25 19.86
water 30 --- 34,632 24,388 3.91 1.42 25 18.91
55 betaine 30 --- 395,583 132,449 5.75 2.99 34 16.49
S/T 30 --- 137,211 65,220 16.68 2.1 30 17.46
water 30 --- 96,976 25,855 3.48 3.75 30 11.43
J63-GA PrimeScript 120 40 betaine 30 --- 1,120,560 516,165 8 2.17 22 27.87
S/T 30 --- 929,189 442,893 11.74 2.1 22 25.11
water 30 --- 1,305,601 601,415 10.01 2.17 22 28.33
45 betaine 30 --- 1,368,533 580,968 17.64 2.36 22 18.73
S/T 30 --- 649,456 318,800 15.27 2.04 22 20.76
water 30 --- 739,001 326,532 18.53 2.26 22 19.22
55 betaine 30 --- 1,003,632 173,383 22.31 5.79 29 25.95
S/T 30 --- 1,217,507 303,138 17.41 4.02 30 42.3
water 30 --- 1,987,403 401,085 17.62 4.96 30 25.45
30 40 betaine 30 --- 873,112 428,106 16.26 2.04 27 22.09
S/T 30 --- 930,354 443,523 17.4 2.1 27 20.09
water 30 --- 848,335 426,379 18.12 1.99 27 30.32
45 betaine 30 --- 574,837 269,437 21.96 2.13 28 26.98
S/T 30 --- 881,363 370,124 20.19 2.38 28 23.45
water 30 --- 312,482 151,889 13.84 2.06 28 17.7
55 betaine 30 --- 1,170,345 200,170 12.99 5.85 38 42.86
S/T 30 --- 586,387 127,215 34.81 4.61 30 14.94
water 30 --- 965,347 119,385 9.25 8.09 40 37.31
J64-GA SuperScript II 120 50 betaine 30 30 917,523 355,315 26.38 2.58 25 38.37
25 908,191 371,519 33.06 2.44 25 49.14
20 787,615 320,633 27.46 2.46 25 30.73
15 --- 987,514 348,994 22.04 2.83 25 25.71
45 --- 1,387,713 484,912 33.68 2.86 25 31.38
60 --- 857,554 334,155 39.52 2.57 21 18.69
SuperScript III 120 50 betaine 30 30 722,783 263,787 28.9 2.74 25 55.85
25 834,995 286,939 24.78 2.91 25 45.05
20 1,396,843 391,729 43.93 3.57 25 46.16
15 --- 1,400,473 347,451 37.41 4.03 27 51.23
45 --- 1,102,061 320,173 48.44 3.44 24 45.75
60 --- 776,672 266,856 39.35 2.91 24 55.63
PrimeScript 120 50 betaine 30 30 1,017,950 274,698 34.21 3.71 28 42.12
25 907,559 219,400 47.38 4.14 27 18.09
20 705,686 190,585 31.01 3.7 28 29
15 --- 1,285,912 270,461 42.59 4.75 28 29.72
45 --- 1,143,364 259,958 54.18 4.4 23 20.19
60 --- 1,191,776 196,585 27.24 6.06 26 25.93
Note: The table lists quality indicators for nanoCAGE libraries made with 50 ng of Hep G2 total RNA using different reverse transcriptases and reaction conditions. The libraries were sequenced on an Illumina Genome AnalyzerIIx. The rows in bold show conditions that gave the best results with SuperScript III and PrimeScript.
aExtension time for the semisuppressive PCR and the PCR to add sequencing adapters. Because the analyzer does not efficiently sequence cDNAs >1 kb in mixtures, short incubation times were tested to reduce the size of the amplified cDNAs.
bS/T: sorbitol/trehalose; water: nuclease-free water.
cAfter the RT reaction, the cDNAs were incubated for 5 min at the indicated temperatures before cooling in an ice-water bath (see Steps 8-9). “---” indicates that the cool-down treatment was not applied to these combinations.
dNumber of tags remaining after removal of artifacts, primer dimers, etc., from the raw sequences.
eNumber of unique tags remaining after removal of artifacts, primer dimers, etc., from the raw sequences.
fPercentage of total tags mapped to ribosomal RNA.
gTotal number of tags divided by the number of unique tags.
hNumber of PCR cycles required to amplify the purified cDNAs by semisuppressive PCR (see Step 17).
iConcentration of semisuppressive PCR products after purification (see Step 24).
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  1. doi:10.1101/pdb.tab195559 Cold Spring Harb Protoc 2011: pdb.tab195559-
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