RNA-seq comparing wild-type and MEIS1/2 knockout cells in cardiac differentiation
RNA-seq comparing wild-type and MEIS1/2 knockout cells in cardiac differentiation
Embryoid bodies were collected on the specified day and lysed in Trizol for RNA extraction. RNA was extracted using TRIzol followed by purification using the Purelink RNA Mini Kit (Thermo Fisher Scientific), according to the manufacturer's instructions. Genomic DNA was removed by on-column DNAse digestion. Quality and integrity of the RNA samples were assessed using a 4200 TapeStation (Agilent Technologies) and then libraries generated using the Illumina® Stranded mRNA Prep Ligation kit (Illumina, Inc.) according to the manufacturer's protocol. Briefly, total RNA (typically 0.025-1 µg) was used as input material from which polyadenylated mRNA was purified using poly-T, oligo-attached, magnetic beads. Next, the mRNA was fragmented under elevated temperature and then reverse transcribed into first strand cDNA using random hexamer primers and in the presence of Actinomycin D (thus improving strand specificity whilst mitigating spurious DNA-dependent synthesis). Following removal of the template RNA, second strand cDNA was then synthesized to yield blunt-ended, double-stranded cDNA fragments. Strand specificity was maintained by the incorporation of deoxyuridine triphosphate (dUTP) in place of dTTP to quench the second strand during subsequent amplification. Following a single adenine (A) base addition, adapters with a corresponding, complementary thymine (T) overhang were ligated to the cDNA fragments. Pre-index anchors were then ligated to the ends of the double-stranded cDNA fragments to prepare them for dual indexing. A subsequent PCR amplification step was then used to add the index adapter sequences to create the final cDNA library. The adapter indices enabled the multiplexing of the libraries, which were pooled prior to cluster generation using a cBot instrument.