title: GSM2055327: negative_siRNA_BS_rep2; Mus musculus; Bisulfite-Seq
library_construction_protocol: 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). DNA was extracted with the Quick-gDNA™ MicroPrep kit (Zymo Research, Irvine, CA). Genomic DNA was treated with sodium metabisulfite for bisulfite conversion. Bisulfite specific PCR primers for fifteen target genes (two promoter regions per gene) were designed with Methyl Primer Express V1.0 (Thermo Fisher Scientific, Waltham, MA). Target gene regions were PCR-amplified from the bisulfite-converted DNA with ZymoTaq™ PreMix (Hot start DNA taq polymerase, ZYMO Research). The fifteen genes include Myh6, Myh7, Myh7b, Nppa, Nppb, Tnnc1, Tnni3, Tnnt2, Camta1, Camta2, Cdkn1A, Cdkn1B, Cdkn1C, Mef2c, and Mef2d. PCR products were cleaned with the ZR-96 DNA Clean & Concentrator™-5 kit (ZYMO Research). PCR products from the same genomic DNA sample were pooled at equal concentration ratio. PCR products from the same genomic DNA were made into an indexed sequencing library with the Nextera XT DNA Library Preparation kit and the Nextera XT Index Kit (Illumina). Briefly, amplicon pools were tagmented, PCR-amplified with index primers, and cleaned with AMPure XP beads. The libraries were validated with an Agilent 2200 TapeStation high sensitivity DNA assay and quantitated by a KAPA Library Quantification kit (Illumina platforms; Kapa Biosystems) on a GeneAmp 7300 Real Time PCR (Thermo Fisher Scientific).
attributes.tag: GEO Accession
attributes.value: GSM2055327