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| Status |
Public on Jul 10, 2021 |
| Title |
2169-1_heart_WT |
| Sample type |
SRA |
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| Source name |
whole heart
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| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6J
genotype: wildtype
Sex: male
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| Treatment protocol |
No treatment
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| Extracted molecule |
total RNA |
| Extraction protocol |
To obtain high quality total RNA, the hearts were processed first in the Trizol reagent (ThermoFisher Scientific, Catalog # 15596018), and then by RNeasy mini columns (Qiagen, catalog # 74104) as follows. To avoid sampling bias, the entire hearts were homogenized in Trizol reagent at a weight (mg) to volume (µL) ratio of 1:15. Then, 1 mL of the homogenate was processed following the manufacturer's protocol until the step of phase separation. 0.5 mL of the aqueous phase was then mixed with an equal volume of 70% ethanol for subsequent RNA purification by the RNeasy mini kit. An on-column DNAse (RNase-Free DNase Set, Qiagen catalog # 79254) treatment step was included. The concentration of the RNA was determined by Qubit fluorometric quantitation (Qubit RNA BR Assay Kit, ThermoFisher Scientific, catalog # Q10210). The integrity of the total RNA was determined by a Fragment Analyzer (Advanced Analytical Technologies, Inc). The average RNA Quality Number (RQN) was 8.77 ± 0.07 (Mean ± SEM, n=24).
1 µg of total RNA from each sample was used for RNA sequencing library preparation with the TruSeq® Stranded mRNA Library Prep kit (Illumina, catalog # 20020594). The libraries were barcoded by TruSeq® RNA Unique Dual Indexes (Illumina, catalog # 20022371) and quantified by qPCR on a Bio-Rad CFX Connect Real-Time PCR detection system with the NEBNext Library Quant Kit for Illumina (New England Biolabs, catalog # E7630L). The libraries were normalized to 10 nM, pooled and sequenced.
RNA-seq. RNA sequencing was carried out at Johns Hopkins School of Medicine Genetic Resources Core Facility on a NovaSeq 6000 sequencing system (Illumina) with a S1 flow cell for 200 cycles, generating 100 bp paired-end reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
wildtype
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| Data processing |
#Quality check: fastqc
#Trimming: (tests were done to decide that trimming was not necessary.)
Reads alignment was done with Tophat using mm10_refseq_nopseudo transcriptome index
ClassifyReads: #counts of how many reads are exonic, intronic, intergenic and overlaping a splice junction
Stringtie: assembly the reads into transcripts
Stringtie --merge : # merging transcripts form all samples in one nonredundant file. Automatically filters a number of transfrags that are probably artfifacts. A reference annotation file was used in order to merge novel isoforms and known isoforms and maximize overall assembly quality. # This will be used further on, in the analysis (cuffdiff);
Cuffdiff v2.2.1: # used to find significant changes in transcript expression.
Samtools: samtools index file.bam
IGV: # vizualize alignments and assemblies
Genome_build: Genome version : mm10
Supplementary_files_format_and_content: .xlsx file of FPKM values. One biological sample per column.
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| Submission date |
Jul 10, 2019 |
| Last update date |
Jul 10, 2021 |
| Contact name |
Mark E Anderson |
| E-mail(s) |
mark.anderson@jhmi.edu
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| Organization name |
Johns Hopkins School of Medicine
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| Department |
Department of Medicine
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| Street address |
Rangos 471, 855 N. Wolfe Street
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| City |
Baltimore |
| State/province |
MD |
| ZIP/Postal code |
21205 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE134121 |
Excessive O-GlcNAcylation causes heart failure and sudden death |
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| Relations |
| BioSample |
SAMN12251633 |
| SRA |
SRX6429333 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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