RNA-Seq of hESC-derived spermatogenic cells treated with 5 uM PBB253 biological replicate 2
mRNA was isolated from total RNA using poly-T oligo-attached magnetic beads. mRNA was fragmented using fragmentation buffer. First cDNA strand was synthesized using random hexamer primers and M-MuLV reverse transcriptase. The second strand was generated using DNA polymerase I and RNase H. The double stranded cDNA was purified with AMPure XP beads. Remaining overhands in ds-cDNA were converted to blunt ends using exonuclease and polymerase enzymes. The 3' ends of DNA fragments were adenylated and ligated to the NEBNext Ultra RNALibrary Prep Kit Adaptors with PCR primers complementary to sequences on the flow cell used for sequencing to prepare for hybridization. The fragments were purified using the AMPure XP system. The library was generated using PCR amplification and the products purified using AMPure XP beads. The library was assessed quantitatively using a Qubit 2.0 Fluorometer, Agilent 2100 to detect and confirm the insert size of 250-300 bp and the effective concentration of the library was determined using qPCR.