GSM5943210: WT_MI_D1_1_19; Mus musculus; RNA-Seq
Cardiomyocytes were isolated using Langendorff perfusion from the left ventricle as previously described. Enzymatic dissociation using Langendorff perfusion was performed with 35 mL enzyme solution (type 2 collagenase 1 mg/mL [Worthington], protease type XIV 0.05 mg/mL [Sigma-Aldrich], NaCl 130 mM, KCl 5.4 mM, MgCl2 0.5 mM, NaH2PO4 0.33 mM, D-glucose 22 mM, HEPES 25 mM; pH 7.4) pre-heated to 37 °C, at a rate of 3 mL/min. To neutralize enzymatic activity, fetal bovine serum (FBS) was added to the solution to a final concentration of 0.2% (v/v). After perfusion of digestion buffer, left ventricle was cut according to each sectional region (IR, RR) and cardiomyocytes were subsequently isolated. Cell suspensions were filtered through a 100-μm nylon mesh cell strainer and centrifuged at 100 g for 2 min, after which the supernatant was discarded. To prevent hypercontraction, the cardiomyocytes were resuspended in medium (NaCl 130 mM, KCl 5.4 mM, MgCl2 0.5 mM, NaH2PO4 0.33 mM, D-glucose 22 mM, HEPES 25 mM, FBS 0.2%; pH 7.4) containing a low concentration of calcium (0.1 mM). Rod-shaped live cardiomyocytes (viability of cardiomyocytes was ≥80% for all time points) were collected immediately with a 0.2–2-µL pipette (sample volume, 0.5 µL) and incubated in lysis buffer. Single-cardiomyocyte cDNA libraries were generated using the Smart-seq2 protocol26 and the efﬁciency of reverse transcription was assessed by examining the cycle threshold (Ct) values of control genes (Tnnt2) from quantitative real-time polymerase chain reaction (qRT-PCR) using a CFX96 Real-Time PCR Detection System (Bio-Rad), and the distribution of cDNA fragment lengths was assessed using LabChip GX (Perkin Elmer) and/or TapeStation 2200 (Agilent Technologies). The following primer sets were used for qRT-PCR: Tnnt2 mRNA forward: TCCTGGCAGA GAGGAGGAAG; Tnnt2 mRNA reverse: TGCAGGTCGA ACTTCTCAGC. A Ct value of 25 was set as the threshold. The sequencing libraries were subjected to paired-end 51-bp RNA sequencing on a HiSeq 2510 (Illumina) in rapid mode