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| Status |
Public on Nov 17, 2014 |
| Title |
AntiMir |
| Sample type |
SRA |
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| Source name |
neonatal cardiomyocytes
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| Organism |
Rattus norvegicus |
| Characteristics |
developmental stage: neonate
cell type: cardiomyocyte
treatment: transfected with adeno-associated virus (AAV) containing anti-miR-99/100 and anti-Let-7 shRNAs for knockdown
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| Treatment protocol |
Neonatal cardiomyocytes were isolated and mock-transduced or transduced with anti-miR-99/100 and anti-Let-7a/c containing AAV2/9 for 7 days
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| Growth protocol |
The method has been described before (Eulalio et al., 2012). Briefly, ventricles from neonatal rats (post-natal day 0) were separated from the atria, cut into pieces and then dissociated in calcium and bicarbonate-free Hanks with HEPES (CBFHH) buffer containing 1.75_mg_ml_1 trypsin (BD Difco) and 10_µg_ml_1 DNase II (Sigma), under constant stirring, at room temperature in eight to ten 10-min steps, collecting the supernatant to fetal bovine serum (FBS, Life Technologies) after each step. The collected supernatant was centrifuged to separate the cells, which were then resuspended in Dulbecco's modified Eagle medium 4.5_g_l_1 glucose (DMEM, Life Technologies) supplemented with 5% FBS, 20_µg_ml_1 vitamin B12 (Sigma) and with 100_U_ml_1 of penicillin and 100_µg_ml_1 of streptomycin (Sigma). The collected cells were passed through a cell strainer (40_µm, BD Falcon) and then seeded onto uncoated 100-mm plastic dishes for 2_h at 37_¡C in 5% CO2and humidified atmosphere. The supernatant, composed mostly of CMs, was then collected and pelleted. Cells were resuspended in antibiotic-free media, counted and plated at the appropriate density; cultures of neonatal rat ventricular CMs prepared using this procedure yielded consistently a purity of >90%.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA of all samples was extracted using Rneasy Mini Kit (Qiagen) per manufacturer's instructions
RNA libraries were prepared for sequencing using strand-specific Illumina TruSeq kits per the manufacturer's instructions
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
miR-99/100 and Let-7 knockdown rat neonatal cardiomyocytes
strand specific polyA+ RNA-Seq
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| Data processing |
Illumina Casava v1.8.2 software used for basecalling.
Reads were aligned to the rat genome (rn4) with STAR (v2.2.0c) with default parameters. Only uniquely alignable reads were used for downstream analysis
Reads counts per gene were calculated using HOMER (http://homer.salk.edu/homer/) across annotated gene exons (RefSeq)
Genome_build: rn4
Supplementary_files_format_and_content: The processed data file contains normalized read counts for all RefSeq annotated gene in the rat genome. The read counts were normalized to the total number of reads found in RefSeq exons in each experiment (normalized to the average between the experiments). The columns in the file are: 1) RefSeq ID, 2) chr, 3) gene start, 4) gene end, 5) strand, 6) avg. mRNA length, 7) Number of copies in the genome, 8) annotation, 9) normalized counts for Ctrl, 10) normalized counts for anti-miR-99/100 and Let-7
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| Submission date |
Oct 15, 2014 |
| Last update date |
May 15, 2019 |
| Contact name |
Christopher Benner |
| E-mail(s) |
cbenner@ucsd.edu
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| Organization name |
University of California, San Diego (UCSD)
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| Department |
Medicine
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| Street address |
9500 Gilman Dr. MC 0640
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| City |
La Jolla |
| State/province |
California |
| ZIP/Postal code |
92093-0640 |
| Country |
USA |
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| Platform ID |
GPL18694 |
| Series (2) |
| GSE62387 |
In vivo activation of a conserved microRNA program induces robust mammalian heart regeneration (RNA-Seq) |
| GSE62389 |
In vivo activation of a conserved microRNA program induces robust mammalian heart regeneration |
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| Relations |
| BioSample |
SAMN03112718 |
| SRA |
SRX733649 |
| 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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