GSM8693525: S17_Dorl_ctrol1_Tbx18; Mus musculus; RNA-Seq
The epicardium/subepicardium was dissected as previously described (Lioux et al., 2020) and quickly frozen in separate 1.5 ml Eppendorf tubes on dry ice. For mRNA isolation, samples from at least three different litters were used. As low RNA amount from one single epicardial layer was expected, two dissected epicardia of the same genotype were pooled together. Therefore, the three biological replicates per genotype represent six different hearts pooled by pairs. RNA was then purified from each pool using RNeasy Micro kit (Qiagen 74004). 40 ng of total RNA were used to generate barcoded RNA-seq libraries using the NEBNext Ultra RNA Library preparation kit (New England Biolabs). Briefly, poly A+ RNA was purified using poly-T oligo- attached magnetic beads followed by fragmentation and then first and second cDNA strand synthesis. Next, cDNA 3' ends were adenylated and the adapters were ligated followed by PCR library amplification. Finally, the size of the libraries was checked using the Agilent 2100 Bioanalyzer DNA 1000 chip and their concentration was determined using the Qubit® fluorometer (Life Technologies). Libraries were sequenced on a HiSeq2500 (Illumina) to generate 60 bases single reads and processed with RTA v1.18.66.3. FastQ files for each sample were obtained using bcl2fastq v2.20.0.422 software (Illumina).