NHLBI BXD Aged Heart HFD RNA-Seq (Nov20) TMP Log2

Experiment design

RNA Extraction

Total RNA was extracted using MIRNEASY MINI KIT (Qiagen, Hilden, Germany) according to the manufacturer’s instructions. Approximately 30 mg of left ventricle tissue was 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 > 8.0 were used for library preparation

About tissue

Tissue Harvest 

The animals were sacrificed under saturated isoflurane. Whole heart 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

NHLBI BXD Aged Heart HFD RNA-Seq (Nov20) TMP Log2

About the cases used to generate this set of data:

The study included 66 mice (~12 month) from B6, B6D2F1, D2B6F1, and 31 advanced intercross BXD strains (about 2 mice per strain). All mice were fed on high fat diet (HFD). 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	Sample ID	Case id	Strain	Fuyi_Sex_Final	Sac age/Day	Tissue	Diet
1	A12	042214.06	BXD29	F	369	Heart	High fat diet
2	A13	042214.13	BXD86	F	360	Heart	High fat diet
3	A14	042214.14	BXD86	F	360	Heart	High fat diet
4	A15	042314.15	C57BL/6J	M	363	Heart	High fat diet
5	A16	042314.16	C57BL/6J	M	363	Heart	High fat diet
6	A17	042314.08	B6D2F1	M	404	Heart	High fat diet
7	A18	042314.07	B6D2F1	M	404	Heart	High fat diet
8	A19	042314.05	BXD64	F	366	Heart	High fat diet
9	A20	042314.06	BXD64	F	366	Heart	High fat diet
10	A21	042314.03	BXD100	F	363	Heart	High fat diet
11	A22	042314.04	BXD100	F	363	Heart	High fat diet
12	A23	042314.02	D2B6F1	F	371	Heart	High fat diet
13	A24	042314.01	D2B6F1	F	371	Heart	High fat diet
14	A25	021313.30	BXD87	F	359	Heart	High fat diet
15	A26	021313.29	BXD87	F	359	Heart	High fat diet
16	A27	042915.06	BXD29	F	431	Heart	High fat diet
17	A28	050115.03	BXD65b	F	415	Heart	High fat diet
18	A29	042915.10	BXD32	F	441	Heart	High fat diet
19	A2	042415.14	BXD102	F	353	Heart	High fat diet
20	A30	050115.04	BXD65b	F	415	Heart	High fat diet
21	A31	121515.16	BXD45	F	350	Heart	High fat diet
22	A32	012615.22	BXD50	F	361	Heart	High fat diet
23	A33	012615.21	BXD50	F	361	Heart	High fat diet
24	A34	121515.17	BXD45	F	350	Heart	High fat diet
25	A36	012615.13	BXD44	F	379	Heart	High fat diet
26	A37	012615.24	BXD75	F	367	Heart	High fat diet
27	A3	090512.12	BXD77	F	353	Heart	High fat diet
28	A40	012615.23	BXD75	F	367	Heart	High fat diet
29	A41	042915.16	BXD48a	F	436	Heart	High fat diet
30	A42	042915.15	BXD48a	F	436	Heart	High fat diet
31	A43	111414.15	BXD102	F	351	Heart	High fat diet
32	A44	012615.12	BXD44	F	379	Heart	High fat diet
33	A45	121214.12	BXD73	F	361	Heart	High fat diet
34	A49	121214.14	BXD40	F	358	Heart	High fat diet
35	A4	090512.11	BXD77	F	353	Heart	High fat diet
36	A50	010614.21	BXD90	F	343	Heart	High fat diet
37	A51	121214.04	BXD66	F	367	Heart	High fat diet
38	A52	121214.13	BXD40	F	358	Heart	High fat diet
39	A53	121214.11	BXD73	F	361	Heart	High fat diet
40	A54	021213.24	BXD84	F	343	Heart	High fat diet
41	A55	021213.18	BXD69	F	347	Heart	High fat diet
42	A56	021213.17	BXD69	F	347	Heart	High fat diet
43	A57	021213.16	BXD62	F	343	Heart	High fat diet
44	A58	021213.15	BXD62	F	343	Heart	High fat diet
45	A59	021113.11	BXD51	F	357	Heart	High fat diet
46	A60	010614.14	BXD65	F	367	Heart	High fat diet
47	A61	010614.13	BXD65	F	367	Heart	High fat diet
48	A62	021213.23	BXD84	F	343	Heart	High fat diet
49	A63	090514.03	BXD43	F	367	Heart	High fat diet
50	A64	090514.04	BXD43	F	367	Heart	High fat diet
51	A65	010614.22	BXD90	F	343	Heart	High fat diet
52	A66	010614.17	BXD73b	F	385	Heart	High fat diet
53	A68	121214.22	BXD39	F	350	Heart	High fat diet
54	A69	121214.21	BXD39	F	350	Heart	High fat diet
55	A6	090512.08	BXD66	F	359	Heart	High fat diet
56	A70	121214.18	BXD70	F	357	Heart	High fat diet
57	A71	121214.17	BXD70	F	357	Heart	High fat diet
58	A72	021313.33	BXD98	F	360	Heart	High fat diet
59	A73	021313.34	BXD98	F	360	Heart	High fat diet
60	A74	092216.09	BXD27	F	387	Heart	High fat diet
61	A75	092216.10	BXD27	F	387	Heart	High fat diet
62	A77	121615.02	BXD61	F	364	Heart	High fat diet
63	A78	121615.01	BXD61	F	364	Heart	High fat diet
64	A7	090512.01	BXD9	F	347	Heart	High fat diet
65	A8	090512.02	BXD9	F	349	Heart	High fat diet
66	A97	050115.11	BXD73b	F	474	Heart	High fat diet