Illumina NovaSeq 6000 paired end sequencing; RNA-seq of fetal lamb heart treated with coiled implantation in left atrium against untreated controls
RNA-seq of fetal lamb heart treated with coiled implantation in left atrium against untreated controls
Under maternal general anesthesia and after fetal echocardiography, fetal lambs 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. 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. RNA was extracted using the miRNeasy Mini Kit (QIAGEN). RNA-Seq library preparation was performed following the TruSeq Stranded Total RNA Library Preparation protocol (Illumina, USA) with the Ribo-Zero Gold rRNA removal kit. Briefly, 600 ng of total RNA was used as the input material and submitted to rRNA depletion using biotinylated beads that contain rRNA-targeting-specific oligos. rRNA-depleted RNA samples were fragmented into ranges of 200-300-bases for 4 minutes at 94°C and converted to double stranded cDNA, end-repaired and adenylated at the 3' to create an overhang A to allow for ligation of Illumina adapters with an overhang T. Library fragments were amplified under the following conditions: initial denaturation at 98°C for 10 seconds, followed by 12 cycles of 98°C for 10 seconds, 60°C for 30 seconds and 72°C for 30 seconds, and finally an extension step for 5 minutes at 72°C. At the amplification step, each sample was amplified with a different barcoded adapter to allow for multiplex sequencing.