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Sample GSM4551657

Query DataSets for GSM4551657

Status

Public on Nov 04, 2020

Title

Myo_GFP_1

Sample type

SRA

Source name

Enzyme-dissociated cells

Organism

Mus musculus

Characteristics

cell type: Cardiomyocyte
Sex: male

Treatment protocol

8-week-old adult CD-1 male mice were housed in standard conditions with ad libitum access to normal chow and water. The mice were anaesthetised in a stinger box with 2% isoflurane (Bayer), then intubated and ventilated with 0.25 L/min oxygen with a tidal volume of 250 μL and a respiration rate = 133 strokes/min (Minivent, Harvard Apparatus). Mice were positioned on a heated surgical mat to maintain body temperature. Once fully anaesthetised, the ventrolateral left chest was shaved, and a lateral thoracotomy was performed at the 4th intercostal space. The pericardium was separated using fine nosed tweezers. A 7-0 prolene suture was used to permanently ligate the left anterior descending coronary artery. MI was verified by observing blanching of the myocardium. A Hamilton syringe with a 30-gauge needle was used to inject 1x1011 AAV particles (i.e. caYAP, caBCAT or GFP control). Viruses were diluted in sterile PBS to a final injection volume of 20 μL for all injection groups; 4 separate injections of 5 μL were injected around the infarct penumbra. Following injection, the chest wall was closed using 4-0 silk suture and the overlying skin sutured with 6-0 prolene suture. The mice were supplied with a s.c. injection of buprenorphine (0.1 mg/kg) and carprofen (10 mg/kg) and allowed to recover. Starting from the day of the surgery, mice received BrdU injections (100 mg/kg, i.p.) every second day for 6 doses.

Extracted molecule

total RNA

Extraction protocol

Adult CD-1 (ICR) mice were anaesthetized with an i.p injection of ketamine (100 mg/kg) and Xylazil (12.5 mg/kg) 3 days 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 KH2PO4, 0.6 mmol/l Na2HPO4, 1.2 mmol/l MgSO4•7H2O, 4.6 mmol/l NaHCO3, 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 was 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 30xg 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 500xg for 5 minutes and FACS was performed.
For enzymatically isolated cells, ribosomal RNA was depleted with Ribo Zero Gold (Illumina), RNA quality ascertained using a MultiNA bioanalyser (Shimadzu) and cDNA generated with SuperScript II Reverse Transcriptase (ThermoFisher). Libraries were created with TruSeq Stranded Total RNA kits (Illumina) and read with HiSeq SR Cluster v4 kit (Illumina) on a HiSeq 2500 sequencer. Each sample contained ~45 million 50-bp single-end reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 4000

Description

Ribo-depleted RNA
20200506_MIP56.BCATvsGFP_CPM
20200506_MIP56.BCATvsGFP
20200506_MIP56.BCATvsGFP_Countmatrix

Data processing

FASTQ files were quality trimmed with Trimmomatic
Reads were mapped to hg38 or mm10 using RNA STAR
Count matrix was calculated using Htseq tools
EdgeR was used for all comparisons
Genome_build: GRCh38/hg38 or GRCm38/mm10

Submission date

May 13, 2020

Last update date

Nov 04, 2020

Contact name

James Hudson

E-mail(s)

James.Hudson@QIMRBerghofer.edu.au

Organization name

QIMR Berghofer Medical Research Institute

Lab

Cardiac Bioengineering Laboratory