GSM5034712: cardiomyocytes tansfected with siHoxa5, repeat3; Mus musculus; RNA-Seq
Adult mouse  cardiomyocytes  were isolated and plated as follows: In brief, heart was exposed first, and EDTA buffer is injected steadily into the right ventricle after cutting of descending artery. The heart then was removed from the mice , perfused with EDTA buffer and collagenase buffer. After digestion completed, cardiac myocytes may be collected by gravity settling, then treated with calcium re-introduction. Adult mouse cardiac myocytes were plated on laminated culture dishes(Nest, 801002) as described. One hours post-plating, myocytes are washed once with pre-equilibrated culture media, and then replaced with the M199 Culture media（gibco,12340-030）. Sequencing libraries were generated using the TruSeq RNA Sample Preparation Kit (Illumina, San Diego, CA, USA). Briefly, mRNA was purified from total RNA using poly-T oligo-attached magnetic beads. Fragmentation was carried out using divalent cations under elevated temperature in an Illumina proprietary fragmentation buffer. First strand cDNA was synthesize dusing random oligonucleotides and SuperScript II. Second strand cDNA synthesis was subsequently performed using DNA Polymerase I and RNase H. Remaining overhangs were converted into blunt ends via exonuclease/polymerase activities and the enzymes were removed. After adenylation of the 3′ ends of the DNA fragments, Illumina PE adapter oligonucleotides were ligated to prepare for hybridization. To select cDNA fragments of the preferred 380 bp in length, the library fragments were purified using the AMPure XP system (Beckman Coulter,Beverly, CA, USA). DNA fragments with ligated adaptor molecules on both ends were selectively enriched using Illumina PCR Primer Cocktail in a 15 cycle PCR reaction. Products were purified (AMPure XP system) and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system (Agilent). The sequencing library was then sequenced on a NovaSeq 6000 platform (Illumina).
GEO Accession
GSM5034712