title: GSM8218774: B16-WT, 6h, rep2; Mus musculus; RNA-Seq
library_construction_protocol: After qualifying the total RNA samples, purification is performed based on the characteristics of mRNA from different sources. Eukaryotic mRNA, which has a polyA tail at the 3' end, is enriched and purified using magnetic beads with oligo(dT). Fragmentation Buffer is added to the purified mRNA to fragment it into short fragments. Then, using the fragmented RNA as a template, the first-strand cDNA synthesis is achieved through reverse transcription with random primers. The second strand of cDNA is synthesized by adding Second Strand Marking Buffer and Second Strand/End Repair Enzyme Mix. After end repair, addition of base A, and sequencing adapter ligation, the target fragments are recovered by magnetic bead selection and subjected to PCR amplification, thus completing the entire library construction process.  Following library construction, the library is initially quantified using Qubit 3.0 and diluted to 1ng/μl. Subsequently, the insert size of the library is detected using Agilent 2100. Once the insert size meets expectations, accurate quantification of the effective concentration of the library is performed using a Bio-RAD CFX 96 fluorescence quantitative PCR instrument and Bio-RAD KIT iQ SYBR GRN for Q-PCR. This ensures precise quantification of the library's effective concentration (>10 nM) to guarantee library quality.