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