GSM7316679: PYMT cells, BMP4-TGFB2_3; Mus musculus; RNA-Seq
Total RNA was extracted using the Qiagen RNeasy Plus Universal kit following the manufacturer's instructions (Qiagen). Extracted RNA samples were quantified using Qubit 2.0 Fluorometer (Life Technologies), and RNA integrity was checked using Agilent TapeStation 4200 (Agilent Technologies). RNA sequencing libraries were prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina following the manufacturer's instructions (NEB). Briefly, mRNAs were first enriched with Oligo(dT) beads. Enriched mRNAs were fragmented for 15 minutes at 94 °C. First- strand and second-strand cDNAs were subsequently synthesized. cDNA fragments were end-repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by limited-cycle PCR. The sequencing libraries were validated on the Agilent TapeStation (Agilent Technologies), and quantified by using Qubit 2.0 Fluorometer (Invitrogen) as well as by quantitative PCR (KAPA Biosystems). The sequencing libraries were clustered on a flowcell. After clustering, the flowcell was loaded on the Illumina HiSeq instrument (4000 or equivalent) according to the manufacturer's instructions. The samples were sequenced using a 2x150bp Paired-End (PE) configuration. Image analysis and base calling were conducted by Illumina Control Software.