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| Status |
Public on Dec 09, 2016 |
| Title |
Early Treatment Sample 2 |
| Sample type |
SRA |
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| Source name |
kidney, diabetic, early treatment
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| Organism |
Mus musculus |
| Characteristics |
strain/background: D2.B6-Ins2Akita/MatbJ
gender: male
disease status: diabetes
tissue: kidney
treatment: P78-PEDF at 6 wks for 6 wks
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| Treatment protocol |
Diabetic D2.B6-Ins2Akita mice develop hyperglycemia at 3 wks of age and all treatment carried out when the mice were either 6 wks (3 wks hyperglycemic exposure; early stage treatment) or 12 wks (9 wks hyperglycemic exposure; late stage treatment) of age. Only mice with blood glucose levels > 350 mg/dl. The drug tested was P78, a small pigment epithelium derived factor (PEDF) active peptide. Briefly, P78 peptide at a dose of 0.3 μg/g/day or vehicle (phosphate-buffered saline; PBS) was administered by continuous subcutaneous infusion for 6 weeks via the osmotic minipump (no. 2006; Alzet, Durect, Palo Alto, CA), implanted dorsally between the shoulders of the animals. Transcriptome analysis of wild-type and diabetic kidney samples were performed at two stages of diabetes where treatment was initiated at an early stage (6 wks of age; 3 wks hyperglycemic) and late stage (12 wks of age; 9 wks hyperglycemic). Age-, gender-, and weight-matched diabetic Ins2Akita and wild-type (N) non-diabetic controls (D) were used in the study. All animals, including wild-type, were implanted with an osmotic minipump infused with either vehicle (wt and diabetic controls) or the P78 peptide (diabetic mice). Duration of treatment was 6 wks with either peptide or vehicle. One group received treatment at the early stage of diabetes (ET, early treatment) at 6 wks of age and the experiment terminated at 12 wks of age. Treatment in the second group was initiated at late stage diabetes (LT, late treatment) at 12 wks of age and terminated at 18 wks of age. The study was carried out on four groups: 1. Normal (N, wild-type control); 2. Diabetic (control), 3. P78 early treatment (ET), 4. P78 Late treatment (LT). (n=7-13/group).
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| Growth protocol |
The animal studies were approved by the Penn State University College of Medicine Institutional Animal Care and Use Committee, and performed in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All experiments were conducted using male D2.B6-Ins2Akita/MatbJ diabetic animals and their wild type (WT) littermate mice (DBA/2J background), recommended by the Animal Models of Diabetes Complications Consortium (AMDCC) as a model of DN. Mice were provided ad lib access to food and water and were euthanized at the end of the experimental period. Kidney samples for RNA extraction were immediately harvested and frozen in liquid nitrogen at the termination of the experiment.
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| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA-seq, we used 13 kidney tissue samples from wild-type mice, 7 from the diabetic Ins2Akita mice, 8 from early P78 treatment of diabetic Ins2Akita mice, and 7 from late P78 treatment the diabetic Ins2Akita 7 mice. Total RNA was extracted using mirVana kit (Life Technologies) with some modifications. Briefly, a bead mill homogenizer (Bullet Blender, Next Advance) was used to homogenize the tissue using a safe-lock microcentrifuge tube (Eppendorf) and a mass of stainless steel beads (Next Advance, cat# SSB14B) equal to the mass of the tissue. Immediately after two volumes of lysis buffer were added to the tube, samples were mixed in the Bullet Blender for 1 min at a speed of six. Samples were visually inspected to confirm desired homogenization and then incubated at 37 °C for 5 min. The lysis buffer was added up to 0.6 ml, and samples were mixed in the Bullet Blender for 1 min. Optical density values of extracted RNA were measured using NanoDrop to confirm an A260:A280 ratio above 1.9. RNA integration number (RIN) was measured using BioAnalyzer (Agilent) RNA 6000 Nano Kit to confirm RIN above 7.
cDNA libraries were prepared using SureSelect Strand Specific RNA Library Preparation Kit (Agilent) as per the manufacturer's instructions. Briefly, polyA RNA was purified from 1000 ng of total RNA using oligo (dT) beads. Extracted RNA was subjected to fragmentation, reverse transcription, end repair, 3'-end adenylation, adaptor ligation and subsequent PCR amplification and SPRI bead purification (Beckman Coulter). The unique barcode sequences were incorporated in the adaptors for multiplexed high-throughput sequencing. The final product was assessed for its size distribution and concentration using BioAnalyzer High Sensitivity DNA Kit (Agilent) and Kapa Library Quantification Kit (Kapa Biosystems). 12 libraries were pooled and diluted to 2 nM in EB buffer (Qiagen) and then denatured using the Illumina protocol. The denatured libraries were diluted to 10 pM by pre-chilled hybridization buffer and loaded onto TruSeq SR v3 flow cells on an Illumina HiSeq 2500 and run for 50 cycles using a single-read recipe (TruSeq SBS Kit v3) according to the manufacturer's instructions (Illumina).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
JTT42
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| Data processing |
Illumina CASAVA pipeline Version 1.8 was used to extract de-multiplexed sequencing reads.
FastQC (version 0.11.2) (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/) was used to validate the quality of the raw sequence data. Additional quality filtering used FASTX- Toolkit (http://hannonlab.cshl.edu/fastx_toolkit) using a quality score cutoff of 20.
Next, alignment of the filtered reads to the mouse reference genome (mm10) was done using Tophat (version 2.0.9) allowing 2 mismatches.
Picard (version1.102) (https://github.com/broadinstitute/picard) was used to assess proportion of mapped bases to coding, UTR, intronic, and intergenic regions, respectively. Picard was used to find coverage across gene body to determine 5'- or 3'- bias.
FPKM (Fragments Per Kilobase of Exon Per Million Fragments Mapped) values were calculated using Cufflinks Version 2.0.2 as provided with the Ensembl gene annotation (release 78).
Genome_build: mm10 (GRCm38)
Supplementary_files_format_and_content: Transcriptome Cufflinks results in tab-delimited text format.
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| Submission date |
Oct 12, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Anna Salzberg |
| Organization name |
Rubius Therapeutics
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| Street address |
399 Binney St UNIT 300
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| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02139 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE87899 |
Identification of Novel Targets of Diabetic Nephropathy Using RNA-seq |
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| Relations |
| BioSample |
SAMN05901929 |
| SRA |
SRX2241394 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2342538_JTT42.genes.fpkm_tracking.txt.gz |
1.1 Mb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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