Illumina HiSeq 3000 paired end sequencing; RNA-Seq of human fetal hearts at 9, 12 and 16 weeks of gestation
RNA-Seq of human fetal hearts at 9, 12 and 16 weeks of gestation
Human fetal hearts were collected after elective terminations of pregnancy, with informed written parental consent obtained from all subjects. All experimental protocols were approved by local ethical committees (Study Code LREC 08/S1101/1). 3 developmental stages were successfully collected: 9, 12 and 16 weeks of gestation.  Individual pregnancies carried on independently to this study. They were terminated after parental decision. All fetuses used for this study were healthy, as assessed by the clinicians.  Fetuses and their tissues were not treated prior to the experiments.  Whole hearts were homogenised by pestle and mortar in liquid nitrogen. 100 mg of the resulting powder was used to extract RNA. The extraction method was followed as outlined in the BioRad Aurum Total RNA Fatty and Fibrous Tissue Kit (cat no. 732-6830). Briefly, the sample was disrupted in PureZol, after which chloroform was added and the lysate centrifuged. RNA was extracted in the aqueous phase and an equal amount of ethanol was added. The mixture was transferred to a RNA binding column and centrifuged. Low stringency wash solution, DNase I, high stringency wash buffer followed by low stringency wash solution was used to purify the RNA, which was then eluted and stored at -80C until use. All RNA extraction were performed at the same time to eliminate the variable of interest, i.e. gestational age.  Illumina compatible barcoded mRNA sequencing libraries were made from 100 ng of total RNA after polyA selection for mature mRNAs using the TruSeq Stranded mRNA Sample Preparation Kit's high sample protocol (Illumina). Before pooling the libraries were visualised using a Tapestation (Agilent) to determine insert size, while their concentration was calculated using the Picogreen dsDNA quantification assay (Thermofisher Scientific).
Experimental Factor: developmental stage
gestational week 9