GSM7951659: spinal cord, SCI-2; Mus musculus; RNA-Seq
The spinal cord tissues were collected from each group. The sequencing method was performed according to the manufacturer's standard protocol. The total RNA of the samples was first extracted and examined for the concentration and quality of the RNA. Then, the mRNA was enriched and purified with magnetic beads attached to oligo (dT), followed by the addition of Fragmentation Buer to split the mRNA into short fragments, and then this was used as a template, reverse transcription using random primers to synthesize the first strand of cDNA, then synthesize the second strand of cDNA. The product was subjected to end repair, addition of base A, and addition of sequencing junction. The library preparation was completed by screening out the tar-get fragments using magnetic beads and performing PCR amplification. After the library construction was completed, it was quantified and diluted to 1ng/ul. Q-PCR was performed using a fluorescent quantitative PCR instrument (Bio-RAD CFX) to determine the quality of the library, and the quality-qualified library was sequenced using the Illumina platform. RNA libraries were prepared for sequencing using standard lllumina protocols, After qualifying the total RNA samples, purification is conducted based on the characteristics of mRNA from different sources. Eukaryotic mRNA, with a polyA tail structure at the 3' end, is enriched and purified using magnetic beads containing Oligo(dT). In contrast, prokaryotic mRNA lacks this feature, and a reagent kit is used to eliminate rRNA for obtaining more meaningful information. Fragmentation Buffer is added to the purified mRNA to break it into short fragments. Using the fragmented RNA as a template, the reverse transcription process is carried out with random primers, leading to the synthesis of the first cDNA strand. The addition of 2nd Strand Marking Buffer and 2nd Strand/End Repair Enzyme Mix is employed to synthesize the second cDNA strand. Subsequent steps involve end repair, addition of A bases, ligation of sequencing adapters, magnetic bead-based selection to recover the target fragments, and PCR amplification, completing the entire library preparation process.