RNA-Seq of mus musculus: adult male stembrain
Fragmented mRNA was used as a template with random oligonucleotides as primers to synthesize the first strand of cDNA using M-MuLV reverse transcriptase system (M0253L, New England Biolabs). The RNA strand was then degraded with RNaseH (M0297L, New England Biolabs), and the second strand of cDNA was synthesized using DNA polymerase I and dNTPs. The purified double-stranded cDNA underwent end repair, addition of an A-tail, and adapter ligation. AMPure XP beads (A63881, Beckman Coulter) were used to select cDNA fragments approximately 250-300 bp in size, followed by PCR amplification and another round of AMPure XP bead purification of the PCR products, ultimately resulting in the final library. The library was constructed using NEBNext Ultra II RNA Library Prep Kit for Illumina (E7770L, New England Biolabs).