Summary

We created a fetal lamb model of hypoplastic left heart syndrome (HLHS), by implanting coils in the left atrium in mid-gestation. We performed bulk RNA sequencing of left ventricles (LV), right ventricles (RV), ascending aortae (AAo) and pulmonary arteries (PA). Single nucleus RNA sequencing was performed on LV free wall tissue (n = 4 coiled samples, n = 3 controls).

Sample collection protocol

Under maternal general anesthesia and after fetal echocardiography, fetal lambs (Dorset x Rideau Arcott strain; 125 days gestational age) were delivered by hysterotomy. The umbilical cord was clamped and the lamb was euthanized with IV sodium pentobarbital (BimedaMTC, Cambridge, Canada). A median sternotomy was used to harvest the heart, then samples were taken from the LV, RV, AAo and main PA. Tissue was snap frozen with liquid N2 and stored at -80°C.

Nucleic acid extraction protocol

Tissue of the left ventricular free wall (30-50mg per sample) was mechanically dissociated into 1-2mm3 using chilled razor blades (Fisher Scientific). In order to isolate nuclei, the tissue was suspended in lysis buffer (for 5 min) and homogenized using a Dounce homogenizer (Sigma-Aldrich). Intact nuclei were verified by SYBR Green II RNA Gel Stain (10,000X concentrate in DMSO; Thermo Fisher Scientific). Resuspended nuclei were counted and pellets were washed twice in resuspension buffer. The resuspension was filtered with a 40µm Flowmi cell strainer (Sigma-Aldrich), and transferred to a 1.5ml LoBind tube (Sigma-Aldrich). Nuclei were counted again and incubated with DAPI (Sigma-Aldrich), at concentrations suggested by the manufacturer. To exclude debris or nuclei aggregates, fluorescence-activated cell sorting (FACS) was performed with a BD Influx cell sorter (BD Biosciences), gating for DAPI positivity (for 1-1.5hrs). Nuclei were collected and washed with resuspension buffer, and then counted.

Nucleic acid library construction protocol

10x Chromium single cell gene expression technology (3’ v2 10x Genomics) was used to generate single-indexed libraries as per the manufacturer’s protocols.

Nucleic acid sequencing protocol

RNA sequencing was performed on a HiSeq X (LV 1 and 2) or NovaSeq 6000 platform (LV 3 and 4) (Illumina), as per the manufacturer’s recommendations. Sequencing was performed at Princess Margaret Genomics Centre (University Health Network).

Treatment protocol

At gestational age 76 days, coils were implanted in the left heart under continuous ultrasound guidance (Philips HDI 5000), using a completely percutaneous approach under strict aseptic conditions. A 20G needle (6” Quincke; Beckton-Dickinson) was used to deliver platinum coils (0.018” IDC, Boston Scientific) above the mitral valve until the LV appeared underfilled and the ascending aortic flow decreased or became retrograde.

High throughput sequence alignment

Single nucleus sequencing reads were mapped to the de novo reference transcriptome (see Miscellaneous .gtf file: merged.txt), using Cell Ranger v3 (10X). The de novo reference transcriptome was generated by an initial mapping of snRNA-seq reads using Cell Ranger v3 (10X) and a ‘pre-mrna’ (i.e., pre-splicing) reference transcriptome, derived from the O. aries NCBI genome assembly 4.0. Ultimately, we used de novo transcriptome annotations, including extensions of the NCBI genome assembly 4.0 gene coordinates and new open reading frames.

Normalization data transformation protocol

The raw gene × nucleus read counts were normalized using SCTransform, and all seven samples were integrated and clustered using Seurat, as implemented in CReSCENT multi-sample pipeline.