UTHSC BXD Cochlea 2-8 wks RNA-Seq (Feb25) TPM Log2 **

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Experiment design

RNA Extraction

Total RNA was extracted from cochlear tissue using the RNA STAT-60 method (Tel-Test Inc., Amsbio, Abingdon, UK) according to the manufacturer’s instructions. Briefly, tissues were homogenized in RNA STAT-60 (1 mL per 50–100 mg of tissue) using a syringe. Chloroform (0.2 mL) was then added, and the mixture was centrifuged at 12,000×g for 1 hour at 4°C. The aqueous phase was carefully transferred to a clean centrifuge tube and mixed with 0.5 mL of isopropanol, followed by incubation for 1 hour or overnight at −20°C. RNA was pelleted by centrifugation at 12,000×g for 1 hour, washed with 75% ethanol, and dissolved in 50 μL of nuclease-free water (Qiagen, Hilden, Germany). The quality and purity of the RNA were assessed using the Agilent 2100 Bioanalyzer system (Agilent, Santa Clara, CA). Only RNA samples with an RNA integrity number (RIN) of 8 or higher were selected for subsequent analyses.

About cases

The study included 172 mice (16~62 day) from B6, D2, and 40 advanced intercross BXD strains (about 4 mice per strain). 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	CaseID	Strain	Sex	Age (Day)	RIN	Tissue	Batch
1	LL106	BXD180	F	16	8.2	cochlea	batch 3
2	LL107	BXD180	F	16	8.7	cochlea	batch 3
3	LL10	BXD62	M	16	8.7	cochlea	batch 3
4	LL110	BXD40	F	16	8	cochlea	batch 3
5	LL112	BXD40	M	16	8.1	cochlea	batch 3
6	LL114	BXD191	M	16	7.8	cochlea	batch 3
7	LL115	BXD191	F	16	8	cochlea	batch 3
8	LL116	BXD191	F	16	8	cochlea	batch 3
9	LL117	BXD190	M	16	7	cochlea	batch 3
10	LL119	C57BL/6J	F	16	8.3	cochlea	batch 3
11	LL11	C57BL/6J	F	16	7.4	cochlea	batch 3
12	LL122	BXD194	F	16	8.4	cochlea	batch 3
13	LL124	C57BL/6J	F	16	8.5	cochlea	batch 3
14	LL139	DBA/2J	F	16	6.7	cochlea	batch 3
15	LL13	BXD32	F	16	7.9	cochlea	batch 3
16	LL14	BXD32	F	16	7.8	cochlea	batch 3
17	LL16	BXD131	M	16	9.5	cochlea	batch 3
18	LL1	BXD86	M	16	9.2	cochlea	batch 3
19	LL2	BXD86	M	16	9.5	cochlea	batch 3
20	LL3	BXD60	F	16	9.7	cochlea	batch 3
21	LL4	BXD60	M	16	9.2	cochlea	batch 3
22	LL51	BXD29	M	16	7.6	cochlea	batch 3
23	LL52	BXD29	M	16	6.7	cochlea	batch 3
24	LL5	BXD70	F	16	8.1	cochlea	batch 3
25	LL6	BXD70	M	16	9.3	cochlea	batch 3
26	LL7	BXD187	M	16	9.1	cochlea	batch 3
27	LL85	BXD101	F	16	7.6	cochlea	batch 3
28	LL8	BXD187	M	16	8.3	cochlea	batch 3
29	LL9	BXD62	M	16	8.4	cochlea	batch 3
30	S107	BXD151	M	16	6.9	cochlea	batch 2
31	S108	BXD151	M	16	7.4	cochlea	batch 2
32	S110	BXD24	F	16	7.4	cochlea	batch 1
33	S111	BXD24	M	16	7.6	cochlea	batch 1
34	S112	BXD155	F	16	8.2	cochlea	batch 1
35	S116	BXD34	F	16	6.9	cochlea	batch 2
36	S118	BXD34	M	16	7	cochlea	batch 2
37	S121	BXD74	F	16	8.3	cochlea	batch 2
38	S123	BXD74	F	16	8.1	cochlea	batch 1
39	S126	BXD131	F	16	5.6	cochlea	batch 2
40	S13	BXD152	M	16	6.8	cochlea	batch 1
41	S14	BXD152	M	16	6.6	cochlea	batch 1
42	S151	BXD113	0	16	6.7	cochlea	batch 1
43	S155	BXD75	0	16	6.3	cochlea	batch 1
44	S156	BXD75	0	16	7	cochlea	batch 1
45	S158	BXD216	F	16	6.8	cochlea	batch 1
46	S159	BXD216	M	16	6.7	cochlea	batch 1
47	S161	BXD65b	F	16	7	cochlea	batch 1
48	S162	BXD65b	M	16	7.5	cochlea	batch 1
49	S187	BXD65	F	16	6.3	cochlea	batch 1
50	S18	BXD170	M	16	6.5	cochlea	batch 1
51	S193	BXD84	M	16	7.1	cochlea	batch 1
52	S195	BXD84	F	16	7.2	cochlea	batch 1
53	S196	BXD31	M	16	6.7	cochlea	batch 1
54	S197	BXD31	F	16	6.4	cochlea	batch 1
55	S19	BXD170	M	16	6.7	cochlea	batch 1
56	S238	DBA/2J	M	16	6.6	cochlea	batch 2
57	S240	DBA/2J	F	16	6.8	cochlea	batch 2
58	S254	BXD83	M	16	7.8	cochlea	batch 2
59	S255	BXD83	F	16	7.4	cochlea	batch 2
60	S257	BXD73b	M	16	6.4	cochlea	batch 2
61	S258	BXD73b	F	16	7.1	cochlea	batch 2
62	S260	C57BL/6J	F	16	6.8	cochlea	batch 2
63	S261	C57BL/6J	M	16	7	cochlea	batch 2
64	S262	BXD150	F	16	7.4	cochlea	batch 2
65	S263	BXD150	F	16	5.7	cochlea	batch 2
66	S264	BXD155	F	16	8.6	cochlea	batch 2
67	S275	BXD73	F	16	6.2	cochlea	batch 2
68	S277	BXD73	M	16	5.8	cochlea	batch 2
69	S281	BXD5	M	16	6.6	cochlea	batch 2
70	S283	BXD5	M	16	6.4	cochlea	batch 2
71	S285	BXD195	M	16	6.3	cochlea	batch 2
72	S286	BXD195	M	16	7.9	cochlea	batch 2
73	S290	BXD160	F	16	8.9	cochlea	batch 2
74	S291	BXD160	M	16	9.1	cochlea	batch 2
75	S294	BXD75	F	16	9.1	cochlea	batch 2
76	S295	BXD75	M	16	7.8	cochlea	batch 2
77	S296	BXD102	M	16	8.1	cochlea	batch 2
78	S297	BXD102	M	16	7.6	cochlea	batch 2
79	S306	BXD161	M	16	5.6	cochlea	batch 2
80	S307	BXD161	M	16	5.8	cochlea	batch 2
81	S316	BXD15	M	16	7.5	cochlea	batch 2
82	S317	BXD15	M	16	7.7	cochlea	batch 2
83	S32	BXD154	M	16	6.4	cochlea	batch 1
84	S33	BXD154	M	16	6.3	cochlea	batch 1
85	S362	BXD65	M	16	5.9	cochlea	batch 2
86	S380	BXD113	0	16	5.7	cochlea	batch 2
87	S88	BXD79	0	16	6.2	cochlea	batch 2
88	S89	BXD79	0	16	6.1	cochlea	batch 2
89	LL100	C57BL/6J	F	56	6.4	cochlea	batch 3
90	LL102	DBA/2J	F	59	7.4	cochlea	batch 3
91	LL105	BXD187	F	56	6.6	cochlea	batch 3
92	LL108	BXD170	F	56	6.5	cochlea	batch 3
93	LL125	BXD62	F	56	7.6	cochlea	batch 3
94	LL127	BXD83	F	56	8	cochlea	batch 3
95	LL128	BXD83	F	56	8.2	cochlea	batch 3
96	LL133	BXD190	F	59	6.5	cochlea	batch 3
97	LL136	BXD194	F	56	7.8	cochlea	batch 3
98	LL141	BXD151	M	61	7	cochlea	batch 3
99	LL142	BXD151	M	61	5.9	cochlea	batch 3
100	LL18	BXD60	F	56	7.2	cochlea	batch 3
101	LL19	BXD60	F	56	6.9	cochlea	batch 3
102	LL25	C57BL/6J	M	56	7.4	cochlea	batch 3
103	LL29	BXD31	F	56	6.5	cochlea	batch 3
104	LL31	BXD79	F	56	6.7	cochlea	batch 3
105	LL35	BXD155	M	56	7.9	cochlea	batch 3
106	LL36	BXD155	M	56	8.4	cochlea	batch 3
107	LL39	BXD160	M	56	7.2	cochlea	batch 3
108	LL41	DBA/2J	F	56	7.1	cochlea	batch 3
109	LL43	DBA/2J	M	56	7.9	cochlea	batch 3
110	LL44	DBA/2J	M	56	7.2	cochlea	batch 3
111	LL47	BXD34	M	56	7.4	cochlea	batch 3
112	LL50	BXD24	M	56	7.1	cochlea	batch 3
113	LL54	BXD40	F	56	8.4	cochlea	batch 3
114	LL55	BXD40	M	56	6.8	cochlea	batch 3
115	LL57	BXD74	M	58	7.8	cochlea	batch 3
116	LL58	BXD74	F	58	5.7	cochlea	batch 3
117	LL59	BXD83	F	58	6.1	cochlea	batch 3
118	LL63	BXD131	M	58	7.4	cochlea	batch 3
119	LL66	BXD195	F	56	7.5	cochlea	batch 3
120	LL67	BXD73	F	56	7.1	cochlea	batch 3
121	LL68	BXD73	F	56	7.5	cochlea	batch 3
122	LL71	BXD75	F	56	7.5	cochlea	batch 3
123	LL73	BXD15	F	56	7	cochlea	batch 3
124	LL74	BXD15	F	56	6.8	cochlea	batch 3
125	LL75	BXD79	M	56	7	cochlea	batch 3
126	LL80	BXD131	F	57	7.3	cochlea	batch 3
127	LL81	BXD29	F	57	7.2	cochlea	batch 3
128	LL83	BXD62	M	56	7.3	cochlea	batch 3
129	LL88	BXD34	M	62	6.5	cochlea	batch 3
130	LL94	BXD24	M	58	7	cochlea	batch 3
131	LL97	BXD31	F	57	7	cochlea	batch 3
132	LL99	BXD216	M	57	7.1	cochlea	batch 3
133	S131	BXD195	F	60	7.3	cochlea	batch 1
134	S168	BXD73	M	60	5.9	cochlea	batch 1
135	S205	BXD65	F	58	6.3	cochlea	batch 1
136	S207	BXD65	F	58	6.8	cochlea	batch 1
137	S209	BXD29	F	56	7	cochlea	batch 1
138	S215	BXD160	F	61	6.9	cochlea	batch 1
139	S218	BXD180	M	61	6.2	cochlea	batch 1
140	S219	BXD180	M	61	6	cochlea	batch 1
141	S227	BXD187	F	62	7.6	cochlea	batch 2
142	S230	BXD40	F	62	6	cochlea	batch 2
143	S231	BXD40	F	62	6.2	cochlea	batch 2
144	S235	BXD161	F	59	6.7	cochlea	batch 2
145	S237	BXD161	F	59	6.1	cochlea	batch 2
146	S244	BXD152	F	61	7.4	cochlea	batch 2
147	S245	BXD152	M	61	6.4	cochlea	batch 2
148	S248	BXD154	M	61	7.5	cochlea	batch 2
149	S249	BXD154	M	61	6.7	cochlea	batch 2
150	S266	BXD150	M	62	5.7	cochlea	batch 2
151	S268	BXD5	F	61	6.3	cochlea	batch 2
152	S269	BXD5	F	61	6.5	cochlea	batch 2
153	S270	BXD113	F	60	6.7	cochlea	batch 2
154	S272	BXD73b	F	60	6	cochlea	batch 2
155	S273	BXD73b	F	60	6.6	cochlea	batch 2
156	S279	BXD75	F	58	9.7	cochlea	batch 2
157	S280	BXD75	M	58	6.7	cochlea	batch 2
158	S287	BXD102	F	62	7.9	cochlea	batch 2
159	S288	BXD102	F	62	7.9	cochlea	batch 2
160	S299	BXD86	F	62	6.1	cochlea	batch 2
161	S300	BXD86	F	62	6.4	cochlea	batch 2
162	S308	BXD65b	F	60	5.9	cochlea	batch 2
163	S310	BXD70	F	59	7.4	cochlea	batch 2
164	S311	BXD70	F	59	6	cochlea	batch 2
165	S313	BXD84	F	58	6.5	cochlea	batch 2
166	S314	BXD84	F	58	6.5	cochlea	batch 2
167	S320	C57BL/6J	M	55	6.2	cochlea	batch 2
168	S329	BXD170	M	60	5.7	cochlea	batch 2
169	S372	BXD216	M	59	6.3	cochlea	batch 2
170	S376	BXD32	M	58	5.9	cochlea	batch 2
171	S378	BXD101	F	56	6.6	cochlea	batch 2
172	S379	BXD101	F	56	5.6	cochlea	batch 2

About tissue

Tissue Harvest

Animals were sacrificed via rapid cervical dislocation following anesthesia administered through an intraperitoneal (IP) injection of ketamine (40 mg/kg), xylazine (5 mg/kg), and acepromazine (1 mg/kg). Cochlear tissues were promptly isolated from each mouse and preserved in RNAlater (Applied Biosystems, Foster City, CA). The tissues were stored in a single tube at 4°C overnight, followed by freezing at −20°C for at least 24 hours, and subsequently maintained at −80°C for long-term storage.

About data processing

Generation of RNA-seq data

One microgram of RNA was used for cDNA library construction at Novogene using the NEBNext® Ultra RNA Library Prep Kit for Illumina® following the manufacturer’s protocol. Briefly, mRNA was enriched using oligo(dT) beads with two rounds of purification and fragmented randomly with a fragmentation buffer. First-strand cDNA synthesis was performed using random hexamer primers. The second strand was synthesized using a custom second-strand synthesis buffer (Illumina, San Diego, CA, USA), dNTPs, RNase H, and DNA polymerase I to produce double-stranded cDNA (ds cDNA). The ds cDNA underwent terminal repair, poly-adenylation, and sequencing adaptor ligation. After size selection and PCR enrichment, the resulting 250–350 bp insert libraries were quantified using a Qubit 2.0 Fluorometer and validated by quantitative PCR. Size distribution analysis was conducted using an Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). Sequencing of the qualified libraries was performed on an Illumina NovaSeq platform (Illumina, San Diego, CA, USA) with a paired-end 150 run configuration (2×150 bases). Each library generated an average of 40 million raw reads.

Read mapping and normalization

The Mus musculus (mouse) reference genome (GRCm38) and associated gene model annotation files were downloaded from the Ensembl genome browser (https://useast.ensembl.org/). The reference genome index was constructed using HISAT2 v2.0.5, a high-performance alignment tool. Paired-end clean reads were aligned to the reference genome using HISAT2 v2.0.5, which supports spliced alignment and is well-suited for RNA sequencing data. FeatureCounts v1.5.0-p3 was employed to quantify the number of reads mapped to each gene. Gene expression levels were normalized and expressed as Transcripts Per Million (TPM), calculated based on the length of each gene and the number of reads mapped to it. This normalization ensures that the sum of all TPM values at the gene level equals 1,000,000, allowing for accurate cross-sample comparisons. Given that the sequencing data were generated across three batches, batch effects were corrected using the ComBat method. This correction was performed on the log2(TPM + 1)-transformed values, effectively minimizing batch-related variability and ensuring robust downstream analyses.

Notes

This dataset is currently private. For access please contact:

Lu Lu
University of Tennessee Health Science Center
71 S Manassas St, Room 410 TSRB
Memphis, TN 38163 USA
Tel. 901 448-7557
llu@uthsc.edu