Instrument: Illumina NovaSeq 6000
Strategy: RNA-Seq
Source: TRANSCRIPTOMIC SINGLE CELL
Selection: cDNA
Layout: PAIRED
Construction protocol: Cardiomyocytes and noncardiomyocyte were isolated using Langendorff perfusion from the left ventricular free wall. Enzymatic dissociation using Langendorff perfusion was performed with 37 °C pre-warmed 35 mL enzyme solution (collagenase Type II 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) at a rate of 3 mL/min. Enzymes were neutralized with fetal bovine serum (FBS) at a final concentration of 0.2%. Cell suspensions were filtered through a 100 μm nylon mesh cell strainer and centrifuged at 20 g for 3 min. 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 (0.5 mM). Rod-shaped live cardiomyocytes (viability of cardiomyocytes was ≥70%) were collected immediately after isolation from two mice at each condition with a 0.2–2 μL pipette (sample volume, 0.5 μL) and incubated in lysis buffer.The supernatant was collected as non-cardiomyocytes and the cells were continued to lysis process according to the 10X protocol. Sample 731-736,library was performed according to the manufacter's instructions (single cell 3' v3 protocol, 10x Genomics). Briefly, GCs were resuspended in the master mix and loaded together with partitioning oil and gel beads into the chip to generate the gel bead-in-emulsion (GEM). The poly-A RNA from the cell lysate contained in every single GEM was retrotranscripted to cDNA, which contains an Ilumina R1 primer sequence, Unique Molecular Identifier (UMI) and the 10x Barcode. The pooled barcoded cDNA was then cleaned up with Silane DynaBeads, amplified by PCR and the apropiated sized fragments were selected with SPRIselect reagent for subsequent library construction. During the library construction Ilumina R2 primer sequence, paired-end constructs with P5 and P7 sequences and a sample index were added. Other samples, single-cell cDNA libraries were generated using the Smart-seq2 protocol. 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 by examining the dis- tribution of the lengths of cDNA fragments using a 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.