UTHSC BXD Young Adult Eye RNA-Seq (Nov20) TPM Log2

Download datasets and supplementary data files

Experiment design

Total RNA was extracted using Trizol^® reagent (Invitrogen, Grand Island, NY, USA) 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 treated with DNase to avoid DNA contamination, and verified by Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA, USA). RNA with OD260/280 > 1.8 and RIN > 8.0 were used for library preparation

About tissue

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 (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

BXD Eye (2~6 Month) RNA-Seq (Oct30) TPM Log2

The table of samples that are finally used for this study.

Index	RNA ID	case ID	DA_corrected_strain	Fuyi_corrected_Sex	Age (day)	Tissue
1	E508	*060619.13	BXD170	Female	76	Eyeball
2	E509	*060619.14	BXD170	Male	76	Eyeball
3	E510	*060619.16	BXD194	Female	94	Eyeball
4	E511	*060619.17	BXD194	Male	94	Eyeball
5	E512	*060619.19	BXD213	Female	78	Eyeball
6	E513	*060619.20	BXD213	Male	78	Eyeball
7	E514	*060619.22	BXD214	Female	107	Eyeball
8	E515	*060619.23	BXD214	Male	107	Eyeball
9	E516	*060619.02	BXD125	Female	100	Eyeball
10	E517	*060619.03	BXD125	Male	100	Eyeball
11	E518	*060619.05	BXD151	Female	97	Eyeball
12	E519	*060619.06	BXD151	Male	97	Eyeball
13	E520	*060619.08	BXD154	Female	102	Eyeball
14	E521	*060619.09	BXD154	Male	102	Eyeball
15	E522	*060519.06	BXD27	Female	94	Eyeball
16	E523	*060519.07	BXD27	Female	94	Eyeball
17	E524	*060519.09	BXD60	Female	82	Eyeball
18	E525	*060519.10	BXD60	Male	82	Eyeball
19	E526	*060519.11	BXD66	Female	104	Eyeball
20	E527	*060519.12	BXD66	Male	104	Eyeball
21	E528	*060519.13	BXD68	Female	91	Eyeball
22	E529	*060519.16	BXD73a	Female	134	Eyeball
23	E530	*060519.15	BXD68	Male	91	Eyeball
24	E531	*060519.19	BXD78	Female	107	Eyeball
25	E532	*060519.21	BXD79	Female	108	Eyeball
26	E533	*060519.20	BXD78	Male	107	Eyeball
27	E534	*060519.23	BXD79	Male	108	Eyeball
28	E535	*060519.24	BXD124	Female	95	Eyeball
29	E536	*060519.26	BXD124	Male	95	Eyeball
30	E537	*051119.05	BXD216	Female	60	Eyeball
31	E538	*051119.06	BXD216	Male	60	Eyeball
32	E539	42415.13	BXD100	Female	176	Eyeball
33	E550	*061119.09	DBA/2J	Male	172	Eyeball
34	E597	*050118.27	DBA/2J	Female	167	Eyeball
35	E598	*050118.28	DBA/2J	Female	167	Eyeball
36	E599	*050118.29	DBA/2J	Male	167	Eyeball
37	E619	*012420.07	BXD75	Female	95	Eyeball
38	E622	*012420.50	BXD197	Female	128	Eyeball
39	E624	*110918.11	BXD32	Female	153	Eyeball
40	E660	*100819.150	BXD199	Female	116	Eyeball
41	E661	*100819.151	BXD199	Male	117	Eyeball
42	E673	*052920.03	C57BL/6J	Female	121	Eyeball
43	E681	*052920.04	BXD73a	Female	180	Eyeball
44	E683	*052920.07	BXD9	Female	185	Eyeball
45	E684	*052920.08	BXD9	Male	186	Eyeball
46	E691	*072120.06	BXD83	Female	132	Eyeball
47	E692	*072120.07	BXD83	Male	132	Eyeball
48	E693	*072120.08	BXD122	Female	73	Eyeball
49	E695	*072120.11	BXD178	Female	172	Eyeball
50	E696	*072120.10	BXD178	Female	172	Eyeball
51	E697	*072120.12	BXD1	Female	133	Eyeball
52	E698	*072120.13	BXD1	Male	133	Eyeball
53	E700	*072120.15	BXD40	Female	118	Eyeball
54	E701	*072120.16	BXD40	Female	118	Eyeball
55	E702	*072120.17	BXD43	Female	102	Eyeball
56	E703	*072120.18	BXD43	Male	102	Eyeball
57	E704	*072120.20	BXD51	Male	135	Eyeball
58	E705	*072120.19	BXD51	Female	135	Eyeball
59	E707	*072120.22	BXD113	Female	180	Eyeball
60	E708	*072120.23	BXD113	Male	180	Eyeball
61	E710	*072120.25	BXD150	Female	173	Eyeball
62	E711	*072120.26	BXD150	Male	173	Eyeball
63	E714	*072120.29	BXD160	Female	144	Eyeball
64	E715	*072120.30	BXD160	Male	144	Eyeball
65	E716	*072120.31	BXD161	Female	71	Eyeball
66	E717	*072120.32	BXD161	Male	71	Eyeball
67	E719	*072120.34	BXD197	Male	153	Eyeball
68	E720	*072120.35	DBA/2J-Gpnmb	Female	97	Eyeball
69	E721	*072120.36	DBA/2J-Gpnmb	Male	97	Eyeball
70	E724	*072120.39	BXD11	Female	131	Eyeball
71	E725	*072120.40	BXD11	Male	131	Eyeball
72	E729	*072120.44	BXD34	Female	166	Eyeball
73	E730	*072120.45	BXD34	Female	166	Eyeball
74	E731	*072120.46	BXD50	Female	177	Eyeball
75	E732	*072120.47	BXD50	Male	177	Eyeball
76	E736	*072120.51	BXD180	Female	92	Eyeball
77	E743	*072120.58	BXD191	Female	134	Eyeball
78	E744	*072120.59	BXD191	Male	134	Eyeball
79	E747	*072120.62	B6D2F1	Female	129	Eyeball
80	E748	*072120.63	B6D2F1	Male	125	Eyeball
81	E751	*072120.66	BXD2	Female	115	Eyeball
82	E752	*072120.67	BXD2	Male	115	Eyeball
83	E755	*072120.74	BXD187	Female	133	Eyeball
84	E756	*072120.75	BXD187	Male	133	Eyeball
85	E757	*072120.71	BXD169	Male	136	Eyeball
86	E758	*072120.70	BXD169	Female	136	Eyeball
87	E761	*081420.01	BXD128a	Female	144	Eyeball
88	E762	*081420.02	BXD128a	Male	144	Eyeball
89	E763	*081420.03	BXD48a	Female	71	Eyeball
90	E764	*081420.04	BXD48a	Male	71	Eyeball
91	E316	*012420.49	BXD190	Male	148	Eyeball
92	E317	*012420.14	BXD87	Male	120	Eyeball
93	E319	*012420.45	BXD180	Male	109	Eyeball
94	E321	*012420.34	BXD152	Female	131	Eyeball
95	E322	*012420.36	BXD152	Male	131	Eyeball
96	E323	*012420.13	BXD87	Female	120	Eyeball
97	E325	*012420.48	BXD190	Female	148	Eyeball
98	E326	*012420.09	BXD75	Male	95	Eyeball
99	E336	*110918.13	BXD32	Male	153	Eyeball
100	E338	*110918.36	BXD71	Male	174	Eyeball
101	E339	*110918.35	BXD71	Female	174	Eyeball
102	E401	*100819.01	C57BL/6J	Female	110	Eyeball
103	E402	*100819.02	C57BL/6J	Female	110	Eyeball
104	E403	*100819.03	C57BL/6J	Male	110	Eyeball
105	E410	*100819.14	BXD15	Female	94	Eyeball
106	E411	*100819.15	BXD15	Female	94	Eyeball
107	E412	*100819.28	BXD29	Female	68	Eyeball
108	E413	*100819.29	BXD29	Male	68	Eyeball
109	E420	*100819.44	BXD44	Female	152	Eyeball
110	E421	*100819.45	BXD44	Male	152	Eyeball
111	E424	*100819.55	BXD61	Female	120	Eyeball
112	E425	*100819.56	BXD61	Male	120	Eyeball
113	E426	*100819.58	BXD62	Female	99	Eyeball
114	E427	*100819.59	BXD62	Male	99	Eyeball
115	E428	*100819.63	BXD65	Female	121	Eyeball
116	E429	*100819.64	BXD65	Male	121	Eyeball
117	E432	*100819.74	BXD73	Female	154	Eyeball
118	E433	*100819.75	BXD73	Male	154	Eyeball
119	E434	*100819.77	BXD73b	Female	153	Eyeball
120	E435	*100819.78	BXD73b	Male	153	Eyeball
121	E436	*100819.79	BXD75	Female	93	Eyeball
122	E437	*100819.80	BXD75	Male	93	Eyeball
123	E438	*100819.86	BXD86	Female	75	Eyeball
124	E439	*100819.87	BXD86	Male	75	Eyeball
125	E440	*100819.90	BXD89	Female	112	Eyeball
126	E441	*100819.91	BXD89	Male	112	Eyeball
127	E442	*100819.92	BXD90	Female	123	Eyeball
128	E443	*100819.93	BXD90	Male	123	Eyeball
129	E444	*100819.95	BXD101	Female	118	Eyeball
130	E445	*100819.96	BXD101	Male	118	Eyeball
131	E446	*100819.98	BXD102	Female	121	Eyeball
132	E447	*100819.99	BXD102	Male	121	Eyeball
133	E457	*100819.122	BXD155	Female	86	Eyeball
134	E458	*100819.123	BXD155	Male	86	Eyeball
135	E461	*052220.38	BXD84	Female	133	Eyeball
136	E462	*052220.47	BXD195	Male	93	Eyeball
137	E463	*052220.46	BXD195	Female	93	Eyeball
138	E464	*052220.39	BXD84	Male	133	Eyeball
139	E465	*052220.42	BXD31	Female	111	Eyeball
140	E466	*052220.43	BXD31	Male	111	Eyeball
141	E467	*052220.26	BXD24	Female	155	Eyeball
142	E468	*052220.27	BXD24	Male	155	Eyeball
143	E469	*052220.49	BXD218	Male	109	Eyeball
144	E470	*052220.36	BXD65b	Female	110	Eyeball
145	E471	*052220.40	BXD202	Female	92	Eyeball
146	E472	*052220.45	BXD122	Male	87	Eyeball
147	E474	*052220.41	BXD202	Male	92	Eyeball
148	E475	*052220.37	BXD65b	Male	110	Eyeball
149	E476	*052220.33	BXD171	Male	74	Eyeball
150	E477	*052220.34	BXD123	Female	127	Eyeball
151	E479	*052220.32	BXD171	Female	74	Eyeball
152	E480	*052220.35	BXD123	Male	127	Eyeball
153	E481	*052220.30	BXD172	Female	104	Eyeball
154	E491	*052220.28	BXD70	Female	118	Eyeball
155	E492	*052220.29	BXD70	Male	118	Eyeball
156	E505	*052220.31	BXD172	Male	104	Eyeball
157	E507	*052220.48	BXD218	Female	109	Eyeball