title: GSM8434643: RNA-seq of cardiac cells from Myh6-dCas9VPR/Klf15 KO mice 3; Mus musculus; RNA-Seq
library_construction_protocol: Cardiac cells were isolated from whole hearts using a modified Langendorff Perfusion protocol. Hearts were perfused with a buffer containing NaCl, HEPES, KCl, glucose, Na-pyruvate, MgCl2, taurine, and 2,3-butadione monoxime, followed by a buffer supplemented with CaCl2 and Liberase DH. Reactions were stopped with a buffer containing bovine serum albumin and CaCl2. Hearts were minced and homogenized into a single cell suspension. Cells were distributed on Matrigel-coated coverslips for live cell imaging using an Axio Imager M2 with ZEN software. RNA was extracted from isolated cardiac cells using standard protocols. RNA quality was assessed by measuring the RNA integrity number (RIN) using a Fragment Analyzer HS Total RNA Kit (Advanced Analytical Technologies, Inc.). Library preparation for RNA-Seq was performed at the Next Generation Sequencing Facility, Institute of Human Genetics, University Medical Center Göttingen. Cells were distributed on ICELL8 250v Chips. Single alive cells were identified using Hoechst 33342 and propidium iodide staining. Complementary DNA synthesis was performed by oligo-dT priming in a one-step RT-PCR reaction. P5 and P7 indexing primers, Terra Polymerase, and Reaction Buffer were used for library preparation. Transposase enzyme and reaction buffer (Tn5 mixture) were added to each well. Final libraries were amplified, pooled, purified, and size-selected with Agencourt AMPureXP magnetic beads to obtain an average library size of 500 bp. Libraries were sequenced on a HiSeq4000 (Illumina) to obtain ~3·10^5 reads per cell (single-end, 50 bp).