NIAAA BXD Hippocampus CMS RNA-Seq (Oct20) TPM Log2

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Experiment design

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 post‐CMS (final 4 weeks). Alcohol was offered as described above for all periods and experimental groups. During the intervening 7‐week CMS period, control animals were subjected to normal housing and experimental (stress) animals were subjected to CMS.

About cases

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

121

CMS111816_13

BXD77

F

CMS+DID

hippocampus

122

CMS111816_26

BXD34

F

CMS+DID

hippocampus

123

S32315_14

C57BL/6J

F

CMS+DID

hippocampus

124

S32415_09

BXD68

F

CMS+DID

hippocampus

125

CMS030716_05

BXD44

F

DID

hippocampus

126

CMS030716_14

BXD40

F

DID

hippocampus

127

CMS030716_21

BXD68

F

DID

hippocampus

128

CMS030716_24

BXD48a

F

DID

hippocampus

129

CMS030716_28

BXD79

F

DID

hippocampus

130

CMS030716_33

BXD65b

F

DID

hippocampus

131

CMS030916_04

BXD55

F

DID

hippocampus

132

CMS030916_06

BXD65b

F

DID

hippocampus

133

CMS030916_07

BXD60

F

DID

hippocampus

134

CMS030916_10

BXD63

F

DID

hippocampus

135

CMS030916_18

BXD65b

F

DID

hippocampus

136

CMS030916_22

BXD79

F

DID

hippocampus

137

CMS030916_24

BXD63

F

DID

hippocampus

138

CMS030916_29

BXD55

F

DID

hippocampus

139

CMS030916_33

BXD69

F

DID

hippocampus

140

CMS032817_08

BXD73

F

DID

hippocampus

141

CMS032817_24

BXD70

F

DID

hippocampus

142

CMS032917_07

BXD70

F

DID

hippocampus

143

CMS032917_19

BXD78

F

DID

hippocampus

144

CMS032917_21

BXD86

F

DID

hippocampus

145

CMS032917_23

BXD66

F

DID

hippocampus

146

CMS032917_30

BXD78

F

DID

hippocampus

147

CMS032917_31

BXD48

F

DID

hippocampus

148

CMS032917_32

BXD71

F

DID

hippocampus

149

CMS033017_16

BXD62

F

DID

hippocampus

150

CMS033017_29

BXD83

F

DID

hippocampus

151

CMS071216_04

BXD90

F

DID

hippocampus

152

CMS071216_09

BXD77

F

DID

hippocampus

153

CMS071216_16

BXD65

F

DID

hippocampus

154

CMS071216_22

BXD48a

F

DID

hippocampus

155

CMS071316_15

BXD90

F

DID

hippocampus

156

CMS071316_16

BXD50

F

DID

hippocampus

157

CMS071316_27

BXD65

F

DID

hippocampus

158

CMS071316_30

BXD51

F

DID

hippocampus

159

CMS071416_10

BXD44

F

DID

hippocampus

160

CMS071416_21

BXD69

F

DID

hippocampus

161

CMS082817_12

BXD51

F

DID

hippocampus

162

CMS082817_16

BXD101

F

DID

hippocampus

163

CMS082817_22

BXD101

F

DID

hippocampus

164

CMS082817_23

BXD24

F

DID

hippocampus

165

CMS082917_13

BXD40

F

DID

hippocampus

166

CMS082917_22

BXD87

F

DID

hippocampus

167

CMS083017_10

BXD24

F

DID

hippocampus

168

CMS102215_20

BXD73B

F

DID

hippocampus

169

CMS111716_02

BXD75

F

DID

hippocampus

170

CMS111716_05

BXD43

F

DID

hippocampus

171

CMS111716_08

BXD75

F

DID

hippocampus

172

CMS111716_10

BXD32

F

DID

hippocampus

173

CMS111716_16

BXD100

F

DID

hippocampus

174

CMS111716_20

BXD60

F

DID

hippocampus

175

CMS111716_22

BXD83

F

DID

hippocampus

176

CMS111716_23

BXD73b

F

DID

hippocampus

177

CMS111716_25

BXD73a

F

DID

hippocampus

178

CMS111816_01

BXD34

F

DID

hippocampus

179

CMS111816_03

BXD48

F

DID

hippocampus

180

CMS111816_08

BXD43

F

DID

hippocampus

181

CMS111816_11

BXD87

F

DID

hippocampus

182

CMS111816_14

BXD60

F

DID

hippocampus

183

CMS111816_16

BXD73b

F

DID

hippocampus

184

CMS111816_17

BXD77

F

DID

hippocampus

185

CMS111816_20

BXD73a

F

DID

hippocampus

186

CMS111816_28

BXD50

F

DID

hippocampus

187

S32515_12

BXD68

F

DID

hippocampus

188

S32515_16

C57BL/6J

F

DID

hippocampus

189

13118_35

BXD62

F

CTL

hippocampus

190

13118_41

BXD66

F

CTL

hippocampus

191

13118_56

BXD75

F

CTL

hippocampus

192

31218_19

BXD65b

F

CTL

hippocampus

193

31218_3

BXD73a

F

CTL

hippocampus

194

31218_36

BXD78

F

CTL

hippocampus

195

AGE041118_03

C57BL/6J

F

CTL

hippocampus

196

AGE050118_07

BXD55

F

CTL

hippocampus

197

AGE050118_12

BXD73

F

CTL

hippocampus

198

AGE050118_16

BXD77

F

CTL

hippocampus

199

AGE050118_21

BXD86

F

CTL

hippocampus

200

AGE050118_23

BXD86

F

CTL

hippocampus

201

AGE050118_27

DBA/2J

F

CTL

hippocampus

202

AGE061818_02

BXD100

F

CTL

hippocampus

203

BL013118_12

BXD34

F

CTL

hippocampus

204

BL013118_13

BXD34

F

CTL

hippocampus

205

CMS092118_24

C57BL/6J

F

CTL

hippocampus

206

E100118_16

BXD65

F

CTL

hippocampus

207

MT052417_09

BXD40

F

CTL

hippocampus

208

MT052417_17

BXD51

F

CTL

hippocampus

209

S13118_07

BXD24

F

CTL

hippocampus

210

S13118_08

BXD24

F

CTL

hippocampus

211

S13118_15

BXD40

F

CTL

hippocampus

212

S13118_18

BXD43

F

CTL

hippocampus

213

S13118_19

BXD43

F

CTL

hippocampus

214

S13118_23

BXD48

F

CTL

hippocampus

215

S13118_24

BXD48

F

CTL

hippocampus

216

S13118_26

BXD48a

F

CTL

hippocampus

217

S13118_36

BXD62

F

CTL

hippocampus

218

S13118_38

BXD65

F

CTL

hippocampus

219

S13118_42

BXD66

F

CTL

hippocampus

220

S13118_44

BXD68

F

CTL

hippocampus

221

S13118_58

BXD79

F

CTL

hippocampus

222

S13118_64

BXD90

F

CTL

hippocampus

223

S13118_65

BXD90

F

CTL

hippocampus

224

S13118_7

BXD101

F

CTL

hippocampus

225

S31218_01

BXD32

F

CTL

hippocampus

226

S31218_02

BXD32

F

CTL

hippocampus

227

S31218_04

BXD44

F

CTL

hippocampus

228

S31218_15

BXD63

F

CTL

hippocampus

229

S31218_16

BXD63

F

CTL

hippocampus

230

S31218_18

BXD65b

F

CTL

hippocampus

231

S31218_1

BXD51

F

CTL

hippocampus

232

S31218_21

BXD69

F

CTL

hippocampus

233

S31218_22

BXD69

F

CTL

hippocampus

234

S31218_24

BXD70

F

CTL

hippocampus

235

S31218_25

BXD70

F

CTL

hippocampus

236

S31218_27

BXD71

F

CTL

hippocampus

237

S31218_28

BXD71

F

CTL

hippocampus

238

S31218_31

BXD73a

F

CTL

hippocampus

239

S31218_35

BXD78

F

CTL

hippocampus

240

S32718_01

DBA/2J

F

CTL

hippocampus

241

S32718_03

DBA/2J

F

CTL

hippocampus

About tissue

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). 

About data processing

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).

Specifics of this data set

NIAAA BXD Hippocampus CMS RNA-Seq (Oct20) TPM Log2