GSM6310520: Bend3, rBMP4, +inhibitor, 8 hr; Mus musculus; RNA-Seq
Total RNA was extracted from micro punched samples using the RNeasy Mini kit. 800ng of total RNA was used for the construction of sequencing libraries. After the QC procedures, mRNA from eukaryotic organisms is enriched from total RNA using oligo(dT) beads. For prokaryotic samples, rRNA is removed using a specialized kit that leaves the mRNA. The mRNA from either eukaryotic or prokaryotic sources then fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamers and reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) is added, with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick-translation and AMPure XP beads is used to purify the cDNA. The final cDNA library is ready after a round of purification, terminal repair, A-tailing, ligation of sequencing adapters, size selection and PCR enrichment. Library concentration was first quantified using a Qubit 2.0 fluorometer (Life Technologies), and then diluted to 1 ng/ul before checking insert size on an Agilent 2100 and quantifying to greater accuracy by quantitative PCR (Q-PCR) (library activity >2 nM).