GSM2053534: DNMT3b_siRNA_rep2; Mus musculus; RNA-Seq
At 72 h after transfection, cells were released with Accutase (Innovative Cell Technologies, San Diego, CA) and dissociated with Accumax (Innovative Cell Technologies). To sort cardiomyocytes, dissociated cells were stained with anti-VCAM1 antibody conjugated with allophycocyanin (APC; BioLegend, San Diego, CA) and magnetically sorted using anti-APC microbeads and magnetic assisted cell sorting (MACS) columns (Miltenyi Biotec, Bergisch Gladbach, Germany). Total RNA was isolated from the sorted cardiomyocytes with the RNAqueous®-Micro Total RNA Isolation Kit (Thermo Fisher Scientific, Waltham, MA). mRNA was isolated from total RNA using NEXTflex™ Poly(A) Beads (Bioo Scientific, Austin, TX). The mRNA was quantitated using the Qubit RNA assay and Qubit 2.0 Flourometer (Thermo Fisher Scientific), as well as a 2200 TapeStation (Pico mRNA; Agilent Technologies). Sequencing libraries were prepared with the NEBNext® mRNA Library Prep Master Mix Set for Illumina (NEB, Ipswich, MA). Briefly, purified mRNA (50-250 ng) was fragmented to ~200 nucleotide mRNA prior to generating first and second strand cDNA. The resulting cDNA was prepared for sequencing by blunt end repair, dA-tailing, adaptor ligation using the NEBNext Multiplex Oligos for Illumina (NEB), size selection using Agencourt AMPure XP Beads (Beckman Coulter, Brea, CA), and PCR enrichment of adaptor ligated cDNA. Library quality was assessed using the Agilent 2200 TapeStation (high sensitivity DNA assay). The concentration of each library was quantitated by a KAPA Library Quantification kit (Illumina platforms; Kapa Biosystems, Woburn, MA) on a GeneAmp 7300 Real Time PCR (Thermo Fisher Scientific).
GEO Accession
GSM2053534