NHLBI BXD Young Adult Heart CD CMS 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 Young Adult Heart CD CMS RNA-Seq (Nov20) TMP Log2

About the cases used to generate this set of data:

The study included 64 mice (~6 month) from B6, D2, and 32 advanced intercross BXD strains (about 2 mice per strain). All mice were fed on chow diet (CD) and treated with chronic mild stress (CMS). All procedures were approved by the UTHSC Institutional Animal Care and Use Committee.

 

Chronic Mild Stress (CMS)

During a period of 7 weeks, mice received 2 disturbances per day. 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.

 

 

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	Treatment
1	C100	090215.15	BXD68	F	185	Heart	Chow diet	CMS
2	C101	090215.16	BXD48	F	171	Heart	Chow diet	CMS
3	C102	090215.20	BXD79	F	162	Heart	Chow diet	CMS
4	C103	090215.21	BXD63	F	169	Heart	Chow diet	CMS
5	C104	090215.24	BXD32	F	188	Heart	Chow diet	CMS
6	C105	090215.28	BXD63	F	169	Heart	Chow diet	CMS
7	C10	CMS102816.14	BXD77	F	124	Heart	Chow diet	CMS
8	C112	CMS032917.33	BXD70	F	162	Heart	Chow diet	CMS
9	C118	CMS082917.05	BXD77	F	196	Heart	Chow diet	CMS
10	C11	CMS102816.15	BXD73b	F	163	Heart	Chow diet	CMS
11	C13	CMS102816.17	BXD100	F	139	Heart	Chow diet	CMS
12	C14	CMS102816.18	BXD43	F	155	Heart	Chow diet	CMS
13	C15	CMS102816.19	BXD83	F	168	Heart	Chow diet	CMS
14	C17	CMS102816.21	BXD65	F	163	Heart	Chow diet	CMS
15	C19	CMS102816.05	DBA/2J	F	156	Heart	Chow diet	CMS
16	C1	CMS082817.05	BXD48	F	174	Heart	Chow diet	CMS
17	C21	CMS102816.07	DBA/2J	F	156	Heart	Chow diet	CMS
18	C23	CMS102816.09	C57BL/6J	F	159	Heart	Chow diet	CMS
19	C25	CMS102816.11	BXD100	F	139	Heart	Chow diet	CMS
20	C28	CMS102716.16	BXD77	F	123	Heart	Chow diet	CMS
21	C2	CMS083017.19	BXD24	F	171	Heart	Chow diet	CMS
22	C30	CMS102716.19	BXD73a	F	121	Heart	Chow diet	CMS
23	C31	CMS102716.20	BXD50	F	161	Heart	Chow diet	CMS
24	C32	CMS102716.01	BXD83	F	167	Heart	Chow diet	CMS
25	C34	CMS102716.10	BXD43	F	144	Heart	Chow diet	CMS
26	C35	CMS102716.12	BXD62	F	144	Heart	Chow diet	CMS
27	C36	CMS102716.03	BXD73a	F	121	Heart	Chow diet	CMS
28	C38	CMS102716.06	BXD34	M	157	Heart	Chow diet	CMS
29	C39	CMS102716.07	BXD24	F	157	Heart	Chow diet	CMS
30	C3	CMS083017.18	BXD87	F	174	Heart	Chow diet	CMS
31	C40	CMS102716.08	BXD60	F	155	Heart	Chow diet	CMS
32	C41	CMS102716.09	BXD60	F	142	Heart	Chow diet	CMS
33	C44	CMS033017.25	BXD78	F	163	Heart	Chow diet	CMS
34	C45	CMS032817.28	BXD66	F	168	Heart	Chow diet	CMS
35	C46	CMS032817.23	BXD86	F	179	Heart	Chow diet	CMS
36	C47	CMS082817.24	BXD51	F	186	Heart	Chow diet	CMS
37	C48	CMS082817.14	BXD65	F	182	Heart	Chow diet	CMS
38	C49	CMS082817.07	BXD44	F	202	Heart	Chow diet	CMS
39	C56	CMS102716.13	BXD77	F	151	Heart	Chow diet	CMS
40	C60	CMS032917.33	BXD70	F	162	Heart	Chow diet	CMS
41	C61	CMS032917.29	BXD71	F	162	Heart	Chow diet	CMS
42	C62	CMS032917.26	BXD87	F	173	Heart	Chow diet	CMS
43	C63	CMS032917.22	BXD71	F	170	Heart	Chow diet	CMS
44	C64	CMS083017.07	BXD69	F	202	Heart	Chow diet	CMS
45	C65	CMS082917.19	BXD44	F	182	Heart	Chow diet	CMS
46	C66	CMS030716.03	BXD101	F	196	Heart	Chow diet	CMS
47	C67	CMS030716.06	BXD69	F	193	Heart	Chow diet	CMS
48	C68	CMS030716.12	BXD68	F	166	Heart	Chow diet	CMS
49	C69	CMS030716.13	BXD55	F	166	Heart	Chow diet	CMS
50	C70	CMS030716.18	BXD40	F	182	Heart	Chow diet	CMS
51	C71	CMS030716.19	BXD40	F	182	Heart	Chow diet	CMS
52	C73	CMS030716.32	BXD101	F	196	Heart	Chow diet	CMS
53	C75	CMS030916.11	BXD79	F	167	Heart	Chow diet	CMS
54	C76	CMS030916.14	BXD48a	F	168	Heart	Chow diet	CMS
55	C77	CMS030916.20	BXD65b	F	190	Heart	Chow diet	CMS
56	C79	CMS030916.23	BXD65b	F	182	Heart	Chow diet	CMS
57	C80	CMS030916.28	BXD66	F	162	Heart	Chow diet	CMS
58	C81	CMS030916.32	BXD86	F	193	Heart	Chow diet	CMS
59	C84	CMS102816.03	C57BL/6J	F	159	Heart	Chow diet	CMS
60	C85	CMS102716.11	BXD34	F	157	Heart	Chow diet	CMS
61	C96	090215.02	BXD62	F	137	Heart	Chow diet	CMS
62	C97	090215.05	BXD75	F	180	Heart	Chow diet	CMS
63	C98	090215.06	BXD32	F	188	Heart	Chow diet	CMS
64	C99	090215.09	BXD75	F	180	Heart	Chow diet	CMS