Computational protocol: Wnt inhibition promotes vascular specification of embryonic cardiac progenitors

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Protocol publication

[…] Total RNA was prepared from FACS-sorted cells and quality was checked on an Agilent Technologies 2100 Bioanalyzer. 1 µg high-quality total RNA was used as input to convert mRNA into a library of template molecules for subsequent cluster generation and sequencing using the reagents provided in the Illumina TruSeq RNA Sample Preparation Kit. Following purification of the poly(A)-containing mRNA using poly(T) oligo-attached magnetic beads, the mRNA was fragmented into small pieces using divalent cations under elevated temperature. The cleaved RNA fragments were copied into first-strand cDNA using reverse transcriptase and random primers, followed by second-strand cDNA synthesis using DNA polymerase I and RNase H. These cDNA fragments then went through an end-repair process, the addition of a single ‘A’ base, and then ligation of adapters. The products were purified and enriched by PCR to create the final cDNA library. After quantifying and checking the size and purity of the product, multiplexed DNA libraries were normalized to 10 nM and then two sample libraries were pooled together in equal volumes. 7 pM each pooled DNA library template was amplified on an Illumina cBot instrument, involving immobilization and 3′ extension, bridge amplification, linearization and hybridization, then sequenced on one lane of the Illumina HiSeq2000 sequencer using the pair-end module and generating 2×58 bp reads. The samples HUVEC, CP/CM, NkxEC and nEC were aligned to the human GRCh38 reference assembly (NCBI) using STAR aligner (Dobin et al., 2013) and subsequently genes were counted in htseq (Harada et al., 1999). The raw counts mapping to GRCh38 were subsequently analyzed in R package edgeR and differential expression analysis was performed. After selecting the most variant 910 genes by expression, multidimensional scaling (MDS) plots were prepared among the sample groups, and showed clear differentiation between the four tissue types (A). […]

Pipeline specifications

Software tools STAR, HTSeq, edgeR
Databases HUVEC
Application RNA-seq analysis
Organisms Homo sapiens, Mus musculus