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 (≤4^o 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