The study included 241 females (aged 50 to 90 ± 2 days at the start of the experiment) from B6, D2, and 35 advanced intercross BXD strains (about 2 mice per strain and per condition). Mice were divided into 4 groups of Control (CTL, 53 mice for 31 strains), Chronic Mild Stress (CMS, 64 mice for 32 strains), Drinking in Dark (DID, 64 mice for 35 strains), and CMS+DID (60 mice for 35 strains). Mice in CTL were normally handled, while mice in other groups were singly housed in shoebox cages, with food and water provided ad libitum except when the water was replaced by 20% (v/v) EtOH (see below) for DID and CMS+DID groups (detailed treatments and schemes are showing in the Table and Figure). The ambient temperature was 21 ± 2°C, and the light cycle was 7:00/19:00 for CTL, and 23:00/11:00 the rest groups lights on/off. The animals were weighed weekly to assess health and to provide the basis for calculating the amount of EtOH consumed in grams per kilogram. All procedures were approved by the UTHSC Institutional Animal Care and Use Committee.
+ +The table of samples that are finally used for this study.
+ +|
+ Index + |
+
+ ID + |
+
+ Strain + |
+
+ Sex + |
+
+ Treatment Group + |
+
+ Tissue + |
+
|
+ 1 + |
+
+ CMS030716_12 + |
+
+ BXD68 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 2 + |
+
+ CMS030716_18 + |
+
+ BXD40 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 3 + |
+
+ CMS030716_19 + |
+
+ BXD40 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 4 + |
+
+ CMS030916_02 + |
+
+ BXD101 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 5 + |
+
+ CMS030916_09 + |
+
+ BXD101 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 6 + |
+
+ CMS030916_11 + |
+
+ BXD79 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 7 + |
+
+ CMS030916_14 + |
+
+ BXD48a + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 8 + |
+
+ CMS030916_20 + |
+
+ BXD65b + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 9 + |
+
+ CMS030916_23 + |
+
+ BXD65b + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 10 + |
+
+ CMS030916_32 + |
+
+ BXD86 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 11 + |
+
+ CMS032817_02 + |
+
+ BXD48 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 12 + |
+
+ CMS032817_28 + |
+
+ BXD66 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 13 + |
+
+ CMS032917_08 + |
+
+ BXD48 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 14 + |
+
+ CMS032917_24 + |
+
+ BXD70 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 15 + |
+
+ CMS032917_26 + |
+
+ BXD87 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 16 + |
+
+ CMS032917_33 + |
+
+ BXD70 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 17 + |
+
+ CMS033017_05 + |
+
+ BXD78 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 18 + |
+
+ CMS033017_25 + |
+
+ BXD78 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 19 + |
+
+ CMS061218_12 + |
+
+ BXD87 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 20 + |
+
+ CMS061218_13 + |
+
+ BXD60 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 21 + |
+
+ CMS061218_14 + |
+
+ BXD71 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 22 + |
+
+ CMS071216_17 + |
+
+ BXD24 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 23 + |
+
+ CMS071216_23 + |
+
+ BXD73B + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 24 + |
+
+ CMS071216_25 + |
+
+ BXD48a + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 25 + |
+
+ CMS071316_06 + |
+
+ BXD90 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 26 + |
+
+ CMS071316_08 + |
+
+ BXD66 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 27 + |
+
+ CMS071316_14 + |
+
+ BXD87 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 28 + |
+
+ CMS071316_17 + |
+
+ BXD62 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 29 + |
+
+ CMS071316_18 + |
+
+ BXD65 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 30 + |
+
+ CMS071316_32 + |
+
+ BXD90 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 31 + |
+
+ CMS071416_06 + |
+
+ BXD50 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 32 + |
+
+ CMS082817_07 + |
+
+ BXD44 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 33 + |
+
+ CMS082817_25 + |
+
+ BXD71 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 34 + |
+
+ CMS082917_16 + |
+
+ BXD79 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 35 + |
+
+ CMS083017_07 + |
+
+ BXD69 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 36 + |
+
+ CMS083017_25 + |
+
+ BXD32 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 37 + |
+
+ CMS092018_01 + |
+
+ DBA/2J + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 38 + |
+
+ CMS092018_05 + |
+
+ C57BL/6J + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 39 + |
+
+ CMS092118_08 + |
+
+ C57BL/6J + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 40 + |
+
+ CMS102716_01 + |
+
+ BXD83 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 41 + |
+
+ CMS102716_02 + |
+
+ BXD75 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 42 + |
+
+ CMS102716_03 + |
+
+ BXD73a + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 43 + |
+
+ CMS102716_05 + |
+
+ BXD73a + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 44 + |
+
+ CMS102716_06 + |
+
+ BXD34 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 45 + |
+
+ CMS102716_07 + |
+
+ BXD24 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 46 + |
+
+ CMS102716_08 + |
+
+ BXD60 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 47 + |
+
+ CMS102716_11 + |
+
+ BXD34 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 48 + |
+
+ CMS102716_12 + |
+
+ BXD62 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 49 + |
+
+ CMS102716_14 + |
+
+ BXD83 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 50 + |
+
+ CMS102716_15 + |
+
+ BXD69 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 51 + |
+
+ CMS102716_16 + |
+
+ BXD77 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 52 + |
+
+ CMS102716_20 + |
+
+ BXD50 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 53 + |
+
+ CMS102816_02 + |
+
+ C57BL/6J + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 54 + |
+
+ CMS102816_03 + |
+
+ C57BL/6J + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 55 + |
+
+ CMS102816_04 + |
+
+ DBA/2J + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 56 + |
+
+ CMS102816_06 + |
+
+ DBA/2J + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 57 + |
+
+ CMS102816_10 + |
+
+ C57BL/6J + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 58 + |
+
+ CMS102816_11 + |
+
+ BXD100 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 59 + |
+
+ CMS102816_13 + |
+
+ BXD75 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 60 + |
+
+ CMS102816_14 + |
+
+ BXD77 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 61 + |
+
+ CMS102816_17 + |
+
+ BXD100 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 62 + |
+
+ CMS102816_18 + |
+
+ BXD43 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 63 + |
+
+ CMS102816_20 + |
+
+ BXD32 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 64 + |
+
+ CMS102816_21 + |
+
+ BXD65 + |
+
+ F + |
+
+ CMS + |
+
+ hippocampus + |
+
|
+ 65 + |
+
+ CMS030716_01 + |
+
+ BXD55 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 66 + |
+
+ CMS030716_08 + |
+
+ BXD63 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 67 + |
+
+ CMS030716_09 + |
+
+ BXD65b + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 68 + |
+
+ CMS030716_15 + |
+
+ BXD69 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 69 + |
+
+ CMS030716_27 + |
+
+ BXD86 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 70 + |
+
+ CMS030716_29 + |
+
+ BXD55 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 71 + |
+
+ CMS030716_30 + |
+
+ BXD62 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 72 + |
+
+ CMS030716_31 + |
+
+ BXD65b + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 73 + |
+
+ CMS030916_15 + |
+
+ BXD69 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 74 + |
+
+ CMS030916_16 + |
+
+ BXD68 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 75 + |
+
+ CMS030916_17 + |
+
+ BXD40 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 76 + |
+
+ CMS030916_26 + |
+
+ BXD79 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 77 + |
+
+ CMS032817_10 + |
+
+ BXD87 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 78 + |
+
+ CMS032817_18 + |
+
+ BXD70 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 79 + |
+
+ CMS032817_25 + |
+
+ BXD48 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 80 + |
+
+ CMS032817_27 + |
+
+ BXD83 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 81 + |
+
+ CMS032817_31 + |
+
+ BXD71 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 82 + |
+
+ CMS032817_32 + |
+
+ BXD70 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 83 + |
+
+ CMS032917_15 + |
+
+ BXD86 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 84 + |
+
+ CMS032917_17 + |
+
+ BXD71 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 85 + |
+
+ CMS032917_25 + |
+
+ BXD78 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 86 + |
+
+ CMS033017_04 + |
+
+ BXD78 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 87 + |
+
+ CMS033017_11 + |
+
+ BXD83 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 88 + |
+
+ CMS033017_13 + |
+
+ BXD73 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 89 + |
+
+ CMS071216_01 + |
+
+ BXD50 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 90 + |
+
+ CMS071216_24 + |
+
+ BXD100 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 91 + |
+
+ CMS071316_05 + |
+
+ BXD51 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 92 + |
+
+ CMS071316_20 + |
+
+ BXD65 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 93 + |
+
+ CMS071316_22 + |
+
+ BXD90 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 94 + |
+
+ CMS071316_29 + |
+
+ BXD51 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 95 + |
+
+ CMS071416_01 + |
+
+ BXD100 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 96 + |
+
+ CMS071416_08 + |
+
+ BXD24 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 97 + |
+
+ CMS071416_13 + |
+
+ BXD48a + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 98 + |
+
+ CMS071416_19 + |
+
+ BXD32 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 99 + |
+
+ CMS071416_27 + |
+
+ BXD63 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 100 + |
+
+ CMS082817_02 + |
+
+ BXD48 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 101 + |
+
+ CMS082817_03 + |
+
+ BXD40 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 102 + |
+
+ CMS082817_09 + |
+
+ DBA/2J + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 103 + |
+
+ CMS082817_28 + |
+
+ BXD79 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 104 + |
+
+ CMS082917_07 + |
+
+ BXD43 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 105 + |
+
+ CMS082917_10 + |
+
+ BXD44 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 106 + |
+
+ CMS082917_18 + |
+
+ BXD32 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 107 + |
+
+ CMS082917_21 + |
+
+ BXD24 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 108 + |
+
+ CMS082917_29 + |
+
+ BXD87 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 109 + |
+
+ CMS083017_02 + |
+
+ BXD101 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 110 + |
+
+ CMS083017_03 + |
+
+ BXD66 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 111 + |
+
+ CMS083017_11 + |
+
+ DBA/2J + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 112 + |
+
+ CMS083017_28 + |
+
+ BXD101 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 113 + |
+
+ CMS111716_07 + |
+
+ BXD75 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 114 + |
+
+ CMS111716_11 + |
+
+ BXD73b + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 115 + |
+
+ CMS111716_12 + |
+
+ BXD75 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 116 + |
+
+ CMS111716_14 + |
+
+ BXD73a + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 117 + |
+
+ CMS111716_17 + |
+
+ BXD50 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 118 + |
+
+ CMS111816_05 + |
+
+ BXD77 + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 119 + |
+
+ CMS111816_07 + |
+
+ BXD73b + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
|
+ 120 + |
+
+ CMS111816_12 + |
+
+ BXD73a + |
+
+ F + |
+
+ CMS+DID + |
+
+ hippocampus + |
+
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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+
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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|
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+
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|
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+
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+
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+
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|
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+
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+
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|
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+
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+
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+
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+
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|
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+
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+
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+
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+
|
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+
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+
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+
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+
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+
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+
|
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+
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+
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|
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+
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+
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+
|
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+
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+
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+
|
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+
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+
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|
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+
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|
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+
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+
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+
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+
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+
+ hippocampus + |
+
|
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+
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+
+ BXD73a + |
+
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+
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+
+ hippocampus + |
+
|
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+
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+
+ BXD78 + |
+
+ F + |
+
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+
+ hippocampus + |
+
|
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+
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+
+ DBA/2J + |
+
+ F + |
+
+ CTL + |
+
+ hippocampus + |
+
|
+ 241 + |
+
+ S32718_03 + |
+
+ DBA/2J + |
+
+ F + |
+
+ CTL + |
+
+ hippocampus + |
+
Alcohol consumption was evaluated using the DID method. The protocol calls for 4 consecutive days of testing. Each day, starting on a Tuesday 3 h after lights were turned off, the water bottles were removed from the cages and replaced with 15 ml centrifuge tubes filled with 20% (v/v) ethanol from 95% USP ethanol. On days 1–3 (Tuesday through Thursday) the length of exposure was 2 h and on the 4th day (Friday) the exposure was 4 h. No alcohol was offered in the intervening period (Saturday through Monday). This protocol was repeated weekly for 16 weeks (Table). Tubes were weighed immediately before and after the exposure period. Volume consumed was converted to g/kg body weight of ethanol.
+ +During a period of 7 weeks, mice received 2 disturbances per day (see Figure). These consisted of being exposed to wet bedding for 1 hour, 1 cm of water in the bottom of the cage with no bedding for 1 hour, no bedding for 1 hour, confinement in a 3 × 3 × 3 cm plastic box for 15 minutes, tilted cage at 45º for 1 hour, and exposure to foreign mouse or fox urine odor for 1 hour. The other stressor was a complete phase shift in the light:dark cycle over each weekend. Disruption of the light cycle (lights constantly on) occurred over the weekend (starting Friday 11:00 am) with the light cycle resuming at 11 am on Monday (lights off). The schedule was repeated weekly over the duration of the study.
+ +Treatment Periods
+ +The entire protocol took 16 weeks to complete and was subdivided into 3 periods: baseline (initial 5 weeks), CMS (next 7 weeks), and postâ€CMS (final 4 weeks). Alcohol was offered as described above for all periods and experimental groups. During the intervening 7â€week CMS period, control animals were subjected to normal housing and experimental (stress) animals were subjected to CMS.
diff --git a/general/datasets/Niaaa_bxd_hip_ctl_rnaseq1020/processing.rtf b/general/datasets/Niaaa_bxd_hip_ctl_rnaseq1020/processing.rtf new file mode 100644 index 0000000..7d83d76 --- /dev/null +++ b/general/datasets/Niaaa_bxd_hip_ctl_rnaseq1020/processing.rtf @@ -0,0 +1,9 @@ +Generation of RNA-seq data
+ +1 µg of RNA was used for cDNA library construction at Novogene using an NEBNext® Ultra RNA Library Prep Kit for Illumina® (cat# E7420S, New England Biolabs, Ipswich, MA, USA) according to the manufacturer’s protocol. Briefly, mRNA was enriched using oligo(dT) beads followed by two rounds of purification and fragmented randomly by adding fragmentation buffer. The first strand cDNA was synthesized using random hexamers primer, after which a custom second-strand synthesis buffer (Illumina, San Diego, CA, USA), dNTPs, RNase H and DNA polymerase I were added to generate the second strand (ds cDNA). After a series of terminal repair, poly-adenylation, and sequencing adaptor ligation, the double-stranded cDNA library was completed following size selection and PCR enrichment. The resulting 250-350 bp insert libraries were quantified using a Qubit 2.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) and quantitative PCR. Size distribution was analyzed using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Qualified libraries were sequenced on an Illumina Novaseq Platform (Illumina, San Diego, CA, USA) using a paired-end 150 run (2×150 bases). An average of 40 million raw reads were generated from each library.
+ ++ +
Read mapping and normalization
+ +Mus musculus (mouse) reference genome (GRCm38) and gene model annotation files were downloaded from the Ensembl genome browser (https://useast.ensembl.org/). Indices of the reference genome were built using STAR v2.5.0a and paired-end reads were aligned to the reference genome. STAR used the method of Maximal Mappable Prefix which can generate a precise mapping result for junction reads. FeatureCount v0.6.1 was used to count the number of read mapped to each gene. Transcripts Per Million (TPM) was calculated for each gene based on the length of the gene and reads mapped to that gene. In this normalization, the sum of all TPMs (genes-level) are equal to 1,000,000. The TPM was further rescaled to log2(TPM+1).
diff --git a/general/datasets/Niaaa_bxd_hip_ctl_rnaseq1020/specifics.rtf b/general/datasets/Niaaa_bxd_hip_ctl_rnaseq1020/specifics.rtf new file mode 100644 index 0000000..072040e --- /dev/null +++ b/general/datasets/Niaaa_bxd_hip_ctl_rnaseq1020/specifics.rtf @@ -0,0 +1 @@ +NIAAA BXD Hippocampus CTL RNA-Seq (Oct20) TPM Log2 \ No newline at end of file diff --git a/general/datasets/Niaaa_bxd_hip_ctl_rnaseq1020/tissue.rtf b/general/datasets/Niaaa_bxd_hip_ctl_rnaseq1020/tissue.rtf new file mode 100644 index 0000000..30cdeea --- /dev/null +++ b/general/datasets/Niaaa_bxd_hip_ctl_rnaseq1020/tissue.rtf @@ -0,0 +1,7 @@ +Tissue Harvest and RNA extraction
+ +The animals were sacrificed under saturated isoflurane. Brains from the animals were dissected under a cold plate (≤4o C), and hippocampus were weighted and snap frozen in dry ice bath with isopentane and stored at −80°C until RNA extraction.
+ +RNA Extraction
+ +Total RNA was extracted using Direct-zol RNA Miniprep Plus Kits (Zymo Research, Irvine, CA, USA) according to the manufacturer’s instructions. Approximately 15 mg of hippocampus tissue was added into a 2 ml tube containing 1 ml TRI Reagent® and one 5 mm stainless steel bead (Qiagen, Hilden, Germany). The tissue was homogenized for 2 min in a Tissue Lyser II (Qiagen, Hilden, Germany) with a speed frequency of 30 rpm followed by incubating for 5 min. 0.1 ml of 1‑bromo-3‑chloropropane was add into the homogenate, shaken vigorously for 15 s, and centrifuged for 15 min at 12,000×g at 4 °C. 600 µl upper aqueous was then transferred into a new collection tube containing 600 µl 100% ethanol. The mixture was loaded into a Zymo-Spin IIC column, wash once with RNA Prep Buffer and twice with RNA Wash Buffer. All RNA had been treated with DNase to eliminate possible DNA contamination, and further precipitated with ethanol. RNA purity and integrity were verified by a nanodrop spectrophotometer and an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA).
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