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| Status |
Public on Jul 24, 2017 |
| Title |
rna_P14_rep2 |
| Sample type |
SRA |
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| Source name |
rna_P14
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| Organism |
Mus musculus |
| Characteristics |
developmental stage: P14
strain: CD1
cell type: Isolated cardiomyocytes
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| Growth protocol |
Neonatal CD-1 (ICR) mice were euthanized by decapitation 3 days after surgery and cardiac ventricles were dissected and washed in ADS buffer (116.3 mmol/l NaCl, 20 mmol/l HEPES, 1 mmol/l NaH2PO4, 5.5 mmol/l glucose, 5.36 mmol/l KCl, 0.83 mmol/l MgSO4,) and then cut into small pieces and pooled into groups of 6 ventricles per cell stirrer. Hearts were then digested at 37 ◦ C under constant agitation for 30-40 minutes (or until tissue was fully homogenized). The digestion buffer was made with 200 μg/ml Liberase DH (Roche) in ADS buffer. Myocytes and non-myocytes were then separated by on a Percoll gradient. Cell isolates were layered on Percoll gradients and centrifuged at 2100 xg for 30 minutes. The bottom layer (containing myocytes) and the top layer (containing enriched non-myocytes) were then collected separately and washed twice in ADS. The myocyte fraction was then lysed in Trizol. FACS was then performed on the non-myocyte fraction. Adult CD-1 (ICR) mice were anesthetized with an i.p injection of ketamine (200 mg/kg) at day 3 post-surgery. Hearts were quickly dissected and washed in perfusion buffer (120.4 mmol/l NaCl, 14.7 mmol/l KCl, 0.6 mmol/l KH 2 PO 4 , 0.6 mmol/l Na 2 HPO 4 , 1.2 mmol/l MgSO 4 •7H2O, 4.6 mmol/l NaHCO 3 , 10 mmol/l Na-HEPES, 30 mmol/l taurine, 5.5 mmol/l glucose and 10 mM 2,3-Butanedione 2-monoxime). The aorta was then cannulated with a 21- gauge cannula, secured with 3-0 silk suture and perfused with 37 ◦ C, oxygenated perfusion buffer using a Langendorff apparatus (4ml/min). Once all blood was washed from the heart, digestion buffer (200 μg/ml Liberase DH (Roche) in perfusion buffer) was passed through the heart for ~8 minutes (until the hearts appeared waxy in color and flaccid to the touch). During perfusion, atria and excess tissue were removed. After enzymatic digestion, hearts were minced with fine scissors into small pieces and triturated to release cells. Cell isolates were passed through a 100 μm cell strainer and centrifuged at 30 xg for 3 minutes at room temperature. Supernatant (containing the non-myocyte cells) was collected and the myocyte pellet was washed with perfusion buffer and re-centrifuged. The supernatant was again collected and the purified myocyte pellet was lysed with 1 ml of Trizol (ThermoFisher). The non-myocyte supernatant was centrifuged at 500 xg for 5 minutes and FACS was performed.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Mouse hearts were pooled to obtain enough material for sequencing (P1: 12 hearts/sample, P14 and P56: 6-7 hearts/sample). The samples were homogenized in 15 mL lysis buffer (0.32 M sucrose, 10 mM Tris-HCl (pH = 8), 5 mM CaCl2, 5 mM magnesium acetate, 2 mM EDTA, 0.5 mM EGTA, 1 mM DTT and 1X complete protease inhibitor (all chemical reagents were acquired from Sigma-Aldrich). The heart lysate was combined with another 15 mL lysis buffer and subsequently homogenized for 10-15 strokes using a dounce tissue grinder (Wheaton, Millville, NJ, USA). The cell lysate was sequentially filtered through 70 μM and 40 μM cell strainers (Becton Dickinson, Franklin Lakes, NJ, USA) and centrifuged to pellet nuclei at 1000 x g for 5 mins. Nuclei pellets were then resuspended in 2-4mL sucrose buffer (1 M sucrose, 10 mM Tris-HCl (pH = 8), 5 mM magnesium acetate, 1 mM DTT and 1X complete protease inhibitor) and the suspension was cushioned on top of a 2x volume of the sucrose buffer, followed by centrifugation to pellet nuclei at 1000 xg for 5 mins. Nuclei pellets were then washed once in 1 mL of nuclei storage buffer (NSB, 0.44 M sucrose, 10 mM Tris-HCl (pH = 7.2), 70 mM KCl, 10 mM MgCl2, 1.5 mM spermine and 1x complete protease inhibitor). The washed nuclei pellets were then resuspended in NSB and stained for the cardiomyocyte nuclear specific marker, Pcm1 (1:200, HPA023374, Sigma-Aldrich) at 4oC for 45 minutes. Omitting the primary antibody served as a control for positive staining. Nuclei were washed twice in NSB and stained with secondary antibody conjugated with Alexa Fluor 633 (1:500, ThermoFisher Scientific (Molecular Probes)) or Brilliant Violet 421 (1:400, BioLegend, San Diego, CA, USA) at 4oC for 30 minutes. Nuclei were washed twice in NSB and resuspended in PBS before being processed for FACS. Pcm1 (-/+) nuclei were separated using the BD FACS ARIA cell sorter. Collected Pcm1 (-/+) nuclei were pelleted at 1500 x g for 15 minutes. The purity of nuclei isolations was checked by qPCR of cardiomyocyte enriched genes. Isolated PCM1 nuclei were resuspended in 1mL TRIzol solution and followed by RNA extraction using Direct-zol RNA mini-prep kit (Zymo Research, Irvine, CA, USA), according to the manufacturer’s recommended protocol, with DNase I treatment in the column to remove genomic DNA contamination. RNA was eluted in 30μL 1xTE buffer. Quantity and quality of RNA were examined on MultiNA bioanalyzer (Shimadzu, Tokyo, Japan).
RNA-seq libraries were prepared using NEBNext rRNA Depletion Kit (#6310, NEB, USA) and NEBNext Ultra Directional RNA Library Prep Kit (#E7420, NEB, USA) according to the manufacturer’s recommended protocol. Libraries were validated on MultiNA bioanalyzer and sequenced at a concentration of 13 pM using HiSeq 2500 version 4 reagents (Illumina, SanDiego,CA, USA), generating 100 base pair reads.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
rRNA depleted RNA
cardiomycyte_rna.mx
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| Data processing |
Used HiSeq Control Software (HCS) v2.2.68 and Real Time Analysis (RTA) v1.18.66.3. The bcl2fastq 2.17.1.14 pipeline was used to generate the sequence data
FastX quality trimmer was used to trim bases with phred quality score less than 20. Reads with a length shorter than 20 bp after clipping were discarded.
Reads were mapped to the mouse genome primary assembly (GRCm38) downloaded from Ensembl with STAR (version2.4.0g1) and Ensembl v85 gene annotations.
FeatureCounts v1.4.2 was used to count reads that mapped to the entire gene body with a mapping quality score of 10 or greater. Genes with fewer than 10 reads per sample on average were excluded.
Genome_build: GRCm38
Supplementary_files_format_and_content: Expression count matrix
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| Submission date |
Mar 07, 2017 |
| Last update date |
May 15, 2019 |
| Contact name |
Mark D Ziemann |
| E-mail(s) |
mark.ziemann@gmail.com
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| Organization name |
Deakin University
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| Department |
Life and Environmental Sciences
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| Street address |
75 Pigdons rd
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| City |
Waurn Ponds |
| State/province |
VIC |
| ZIP/Postal code |
3216 |
| Country |
Australia |
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| Platform ID |
GPL17021 |
| Series (2) |
| GSE95762 |
Multi-cellular Transcriptional Profiling Reveals an Epigenetic Barrier to Adult Heart Regeneration [RNA-Seq] |
| GSE95764 |
Multi-cellular Transcriptional Profiling Reveals an Epigenetic Barrier to Adult Heart Regeneration |
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| Relations |
| BioSample |
SAMN06545354 |
| SRA |
SRX2617454 |
| 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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