RNA-Seq of mus musculus: young male lung
After qualifying total RNA samples, purification based on mRNA characteristics from various sources was conducted. Eukaryotic mRNA's 3' end with polyA tail was enriched using magnetic beads with Oligo(dT). Prokaryotic mRNA lacking this feature necessitated rRNA removal using a specific kit. Purified mRNA was fragmented with Fragmentation Buffer. The fragmented RNA served as a template for reverse transcription with random primers for the first cDNA Strand. The second cDNA Strand was synthesized with 2nd Strand Marking Buffer and 2nd strand/End Repair Enzyme Mix. Terminal repair, base A addition, sequencing joint, and magnetic bead screening facilitated target fragment recovery. PCR amplification completed the library preparation.