UTHSC BXD Young Aged Eye RNA-Seq (Apr22) DESeq2 rlog2

Download datasets and supplementary data files

Experiment design

RNA Extraction

Total RNA was extracted using MIRNEASY MINI KIT (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. according to the manufacturer’s instructions. Both left and right eyeballs from one mouse were added into a 2 mL tube containing 700ul QIAzol Lysis Reagent and one 5 mm stainless steel bead (Qiagen, Hilden, Germany). The tissue was homogenized for 2 minutes in a Tissue Lyser II (Qiagen, Hilden, Germany) with a speed frequency of 30 r followed by incubating for 5 minutes.140 µl chloroform was added into the homogenate, shaken vigorously for 15 seconds, and centrifuged for 15 minutes at 12,000 x g at 4 ℃. 280 µl upper aqueous was then transferred into a new collection tube containing 500 µl 100% ethanol. The mixture was loaded into a RNeasy mini‐spin column (Qiagen, Valencia, CA, USA), once with Buffer RWT and twice with Buffer RPE purification. All RNA had been verified by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA with OD260/280 > 1.8 and RIN > 7.0 were used for library preparation

About cases

The study included 187 mice (12~18 month) from B6, D2, D2-Gpmnb, B6D2F1, D2B6F1, and 87 advanced intercross BXD strains (about 2 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	RNA ID	case ID	DA_corrected_strain	Corrected Sex	AgeAtDeath days	Tissue
1	E18	050115.16	BXD75	Female	460	Eyeball
2	E19	050115.17	BXD75	Female	460	Eyeball
3	E21	042715.10	D2B6F1	Female	500	Eyeball
4	E22	050115.19	BXD79	Female	468	Eyeball
5	E31	121515.13	BXD45	Female	507	Eyeball
6	E33	012615.10	BXD45	Female	384	Eyeball
7	E34	012615.04	BXD62	Female	376	Eyeball
8	E40	021213.21	BXD79	Female	378	Eyeball
9	E49	021213.13	BXD60	Female	530	Eyeball
10	E60	101713.01	BXD60	Female	374	Eyeball
11	E62	090612.07	BXD73b	Female	377	Eyeball
12	E63	090612.08	BXD73b	Female	377	Eyeball
13	E65	050912.04	BXD34	Female	419	Eyeball
14	E67	42214.08	BXD40	Female	362	Eyeball
15	E72	42214.04	BXD24	Female	377	Eyeball
16	E75	42214.03	BXD24	Female	377	Eyeball
17	E78	42314.1	B6D2F1	Male	364	Eyeball
18	E82	121615.05	BXD62	Female	525	Eyeball
19	E87	101014.10	BXD95	Female	536	Eyeball
20	E89	121615.22	BXD89	Female	444	Eyeball
21	E90	101014.09	BXD95	Female	536	Eyeball
22	E99	121214.15	BXD70	Female	357	Eyeball
23	E100	121214.16	BXD70	Female	357	Eyeball
24	E124	042214.18	D2B6F1	Male	397	Eyeball
25	E152	050912.04	BXD48a	Female	419	Eyeball
26	E158	101713.02	BXD100	Female	374	Eyeball
27	E224	021313.26	BXD84	Female	385	Eyeball
28	E264	*050318.12	BXD69	Male	387	Eyeball
29	E288	*110918.106	BXD211	Male	404	Eyeball
30	E289	*110918.105	BXD211	Female	404	Eyeball
31	E290	*100218.04	BXD44	Female	530	Eyeball
32	E291	*100218.05	BXD44	Male	530	Eyeball
33	E292	*100218.18	BXD68	Female	516	Eyeball
34	E293	*100218.20	BXD68	Male	516	Eyeball
35	E294	*100218.36	BXD84	Female	516	Eyeball
36	E298	*100218.73	BXD184	Female	512	Eyeball
37	E299	*100218.74	BXD184	Male	512	Eyeball
38	E303	AGE061218.98	BXD177	Female	364	Eyeball
39	E304	AGE061218.108	BXD195	Male	386	Eyeball
40	E306	AGE061218.107	BXD195	Female	386	Eyeball
41	E308	AGE061218.99	BXD177	Male	364	Eyeball
42	E310	AGE061218.90	BXD168	Male	538	Eyeball
43	E311	AGE061218.85	BXD160	Male	390	Eyeball
44	E312	AGE061218.71	BXD150	Male	398	Eyeball
45	E313	AGE061218.22	BXD34	Male	369	Eyeball
46	E314	AGE061218.21	BXD34	Female	369	Eyeball
47	E331	*110918.07	BXD28	Male	366	Eyeball
48	E334	*110918.02	BXD18	Male	452	Eyeball
49	E335	*110918.01	BXD18	Female	452	Eyeball
50	E340	*110918.51	BXD98	Male	359	Eyeball
51	E341	*110918.50	BXD98	Female	359	Eyeball
52	E344	*083019.05	BXD9	Female	373	Eyeball
53	E345	*083019.06	BXD9	Male	373	Eyeball
54	E346	*083019.22	BXD31	Male	391	Eyeball
55	E347	*083019.23	BXD43	Female	391	Eyeball
56	E349	*083019.26	BXD43	Male	367	Eyeball
57	E350	*083019.30	BXD50	Female	408	Eyeball
58	E351	*083019.31	BXD50	Female	408	Eyeball
59	E352	*083019.33	BXD51	Female	368	Eyeball
60	E353	*083019.34	BXD51	Male	368	Eyeball
61	E354	*083019.38	BXD66	Male	430	Eyeball
62	E355	*083019.39	BXD66	Male	430	Eyeball
63	E356	*083019.45	BXD73	Female	451	Eyeball
64	E357	*083019.46	BXD73	Male	451	Eyeball
65	E358	*083019.48	BXD75	Female	369	Eyeball
66	E359	*083019.49	BXD75	Male	369	Eyeball
67	E360	*083019.50	BXD78	Female	432	Eyeball
68	E361	*083019.51	BXD78	Male	432	Eyeball
69	E363	*083019.55	BXD87	Female	444	Eyeball
70	E364	*083019.56	BXD87	Male	444	Eyeball
71	E365	*083019.57	BXD89	Female	446	Eyeball
72	E366	*083019.66	BXD113	Female	366	Eyeball
73	E367	*083019.67	BXD113	Male	366	Eyeball
74	E368	*090119.04	BXD123	Female	441	Eyeball
75	E369	*090119.05	BXD123	Male	441	Eyeball
76	E370	*090119.09	BXD141	Female	370	Eyeball
77	E371	*090119.11	BXD141	Male	370	Eyeball
78	E372	*090119.15	BXD151	Female	439	Eyeball
79	E373	*090119.16	BXD151	Male	439	Eyeball
80	E374	*090119.18	BXD152	Female	374	Eyeball
81	E375	*090119.19	BXD152	Male	374	Eyeball
82	E376	*090119.20	BXD154	Female	370	Eyeball
83	E377	*090119.22	BXD154	Male	370	Eyeball
84	E378	*090119.24	BXD155	Female	379	Eyeball
85	E379	*090119.25	BXD155	Male	379	Eyeball
86	E380	*090119.27	BXD161	Female	376	Eyeball
87	E381	*090119.28	BXD161	Male	376	Eyeball
88	E382	*090119.34	BXD170	Male	438	Eyeball
89	E383	*090119.36	BXD171	Female	370	Eyeball
90	E384	*090119.37	BXD171	Male	370	Eyeball
91	E385	*090119.41	BXD178	Female	493	Eyeball
92	E386	*090119.42	BXD178	Female	493	Eyeball
93	E387	*090119.44	BXD180	Female	375	Eyeball
94	E390	*090119.48	BXD190	Male	374	Eyeball
95	E391	*090119.52	BXD197	Female	433	Eyeball
96	E392	*090119.53	BXD197	Male	433	Eyeball
97	E395	*090119.57	BXD202	Female	427	Eyeball
98	E399	*090119.67	BXD218	Female	412	Eyeball
99	E400	*090119.68	BXD218	Male	412	Eyeball
100	E448	*100819.101	BXD111	Female	373	Eyeball
101	E449	*100819.102	BXD111	Male	373	Eyeball
102	E450	*100819.104	BXD122	Female	383	Eyeball
103	E451	*100819.105	BXD122	Male	383	Eyeball
104	E452	*100819.107	BXD124	Female	366	Eyeball
105	E453	*100819.108	BXD124	Male	366	Eyeball
106	E454	*100819.110	BXD125	Female	376	Eyeball
107	E455	*100819.111	BXD125	Male	376	Eyeball
108	E459	*100819.131	BXD172	Female	375	Eyeball
109	E460	*100819.132	BXD172	Male	375	Eyeball
110	E482	*052220.14	BXD71	Female	396	Eyeball
111	E483	*052220.10	BXD11	Female	379	Eyeball
112	E484	*052220.07	BXD65	Male	528	Eyeball
113	E485	*052220.03	BXD194	Male	396	Eyeball
114	E486	*052220.19	BXD15	Female	415	Eyeball
115	E487	*052220.02	BXD194	Female	396	Eyeball
116	E488	*052220.12	BXD1	Female	429	Eyeball
117	E489	*052220.18	BXD216	Male	401	Eyeball
118	E490	*052220.13	BXD1	Male	429	Eyeball
119	E494	*052220.15	BXD71	Male	396	Eyeball
120	E496	*052220.08	BXD170	Male	360	Eyeball
121	E498	*052220.06	BXD65	Female	528	Eyeball
122	E499	*052220.05	C57BL/6J	Male	424	Eyeball
123	E501	*052220.21	BXD73a	Female	408	Eyeball
124	E502	*052220.11	BXD11	Male	379	Eyeball
125	E503	*052220.22	DBA/2J-Gpnmb	Female	439	Eyeball
126	E504	*052220.20	BXD15	Male	415	Eyeball
127	E541	042915.08	BXD29	Female	478	Eyeball
128	E542	050115.01	BXD65b	Female	415	Eyeball
129	E543	042915.07	BXD29	Female	431	Eyeball
130	E544	042314.12	B6D2F1	Female	364	Eyeball
131	E548	*061119.02	C57BL/6J	Female	392	Eyeball
132	E549	*061119.03	C57BL/6J	Male	392	Eyeball
133	E551	*061119.11	DBA/2J	Female	392	Eyeball
134	E552	*061119.12	DBA/2J	Male	392	Eyeball
135	E554	*061119.15	DBA/2J	Male	392	Eyeball
136	E556	*061119.22	DBA/2J	Male	380	Eyeball
137	E561	*061219.12	BXD27	Female	494	Eyeball
138	E564	*061219.14	BXD27	Male	494	Eyeball
139	E565	*060419.04	BXD32	Female	425	Eyeball
140	E566	*060419.05	BXD32	Male	425	Eyeball
141	E567	*070919.01	BXD86	Female	381	Eyeball
142	E569	*070919.03	BXD86	Male	381	Eyeball
143	E576	*072519.11	BXD191	Male	477	Eyeball
144	E577	*072519.09	BXD190	Male	434	Eyeball
145	E582	*073019.16	BXD216	Male	459	Eyeball
146	E591	*061319.04	BXD48	Female	425	Eyeball
147	E592	*061319.05	BXD48	Male	425	Eyeball
148	E593	*061319.21	BXD61	Male	459	Eyeball
149	E600	*050318.10	BXD69	Female	387	Eyeball
150	E601	*013020.53	BXD102	Male	416	Eyeball
151	E603	*013020.25	BXD65b	Male	505	Eyeball
152	E605	*013020.54	BXD102	Female	416	Eyeball
153	E620	*012420.26	BXD128a	Male	366	Eyeball
154	E625	*110918.54	BXD101	Male	361	Eyeball
155	E626	*110918.53	BXD101	Female	361	Eyeball
156	E631	*083019.14	BXD16	Female	363	Eyeball
157	E635	*083019.35	BXD61	Male	364	Eyeball
158	E638	*083019.60	BXD90	Female	378	Eyeball
159	E639	*083019.62	BXD90	Male	378	Eyeball
160	E662	*100819.153	BXD202	Male	410	Eyeball
161	E663	*100819.157	BXD205	Female	371	Eyeball
162	E664	*100819.158	BXD205	Male	372	Eyeball
163	E665	*100819.163	BXD213	Female	396	Eyeball
164	E666	*100819.164	BXD213	Male	397	Eyeball
165	E667	*100819.168	BXD223	Female	419	Eyeball
166	E668	*100819.169	BXD223	Male	420	Eyeball
167	E672	*052920.10	BXD83	Male	380	Eyeball
168	E675	*052920.13	DBA/2J-Gpnmb	Female	452	Eyeball
169	E679	*052920.12	DBA/2J-Gpnmb	Female	458	Eyeball
170	E680	*052920.01	BXD43	Female	465	Eyeball
171	E682	*052920.02	C57BL/6J	Female	433	Eyeball
172	E685	*052920.09	BXD83	Female	379	Eyeball
173	E699	*072120.14	BXD40	Male	458	Eyeball
174	E709	*072120.24	BXD150	Female	433	Eyeball
175	E712	*072120.27	BXD160	Female	367	Eyeball
176	E726	*072120.41	BXD16	Female	367	Eyeball
177	E733	*072120.48	BXD60	Female	558	Eyeball
178	E735	*072120.50	BXD180	Male	473	Eyeball
179	E738	*072120.53	BXD128a	Female	387	Eyeball
180	E745	*072120.60	BXD199	Female	457	Eyeball
181	E746	*072120.61	BXD199	Male	457	Eyeball
182	E749	*072120.64	BXD2	Female	399	Eyeball
183	E750	*072120.65	BXD2	Male	399	Eyeball
184	E753	*072120.68	BXD169	Female	441	Eyeball
185	E754	*072120.69	BXD169	Male	441	Eyeball
186	E759	*072120.72	BXD187	Female	354	Eyeball
187	E760	*072120.73	BXD187	Male	354	Eyeball

About tissue

Tissue Harvest

The animals were sacrificed under saturated isoflurane. Eyeballs from the animals were dissected and stored at −80°C until RNA extraction.

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 (mm11 Mus_musculus.GRCm39, release 104) and gene model annotation files were downloaded from the Ensembl genome browser (https://useast.ensembl.org/). Rows with no gene symbol name were deleted. Indices of the reference genome were built using STAR version 2.5.2b and paired-end reads were aligned to the reference genome. FeatureCount from package RsubRead, version 1.32.4, was used to count the number of read mapped to each gene. Raw counts were then normalized and log2 transformed using function rlogTransformation from the DESeq2 package (version 1.16.1) and an increment was added to the normalized values to make all values positive.

Notes

UTHSC BXD Young Aged Eye RNA-Seq (Apr22) DESeq2 rlog2