GSM1542461: control hiPSC-CMs at 2 weeks #1; Homo sapiens; RNA-Seq
Total RNA was extracted and quantified using miRNeasy Kit (Qiagen, Venlo, Netherlands) according to the manufacturer’s protocol. An additional DNaseI digestion step was performed to ensure that the samples were not contaminated with genomic DNA. Following the Qiagen RNA sample preparation protocol, 10 ug of total RNA was used to generate index-tagged paired-end cDNA libraries.5 Briefly, mRNAs were purified by polyA enrichment procedure using Dynal Oligo(dT) beads (Life technologies). mRNA fragmentation was performed using RNA Fragmentation Reagents (Life technologies) with incubation at 70oC for 1 min to obtain 200-300b fragments. cDNA was generated using SuperScript Double-Stranded cDNA Synthesis Kit (Life technologies). Illumina sequencing adapters were ligated to cDNA using LigaFast (Promega) and PE Adapter Oligo Mix (Illumina, San Diego, CA). PCR was performed on the adapter-ligated cDNA with 2X Phusion DNA polymerase Master Mix (New England Biolabs, Ipswich, MA) using the following conditions: denaturation 98°C for 30 seconds, followed by 15 cycles of denaturation 98°C for 10 seconds, annealing 65°C for 30 seconds, and extension 72°C for 30 seconds, ending with an extension at 72°C for 5 min. After cDNA libraries were generated using the sample preparation kit, quality of the libraries was checked using Bioanalyzer (Agilent Technologies).