GSM9557805: mBMSCs, Con, rep2; Mus musculus; RNA-Seq
RNA was harvested through standard wordflow. Quality control was conducted by Agilent 2100 bioanalyzer. Using fragmented mRNA as a template and random oligonucleotides as primers, the first strand of cDNA is synthesised within the M-MuLV reverse transcriptase system. Subsequently, the RNA strand is degraded by RNase H, and the second strand of cDNA is synthesised using dNTPs as substrates within the DNA polymerase I system. The purified double-stranded cDNA undergoes end repair, A-tailing, and sequencing adapter ligation. AMPure XP beads are employed to enrich cDNA fragments of approximately 250–300 bp, which are then amplified via PCR. The PCR products are further purified using AMPure XP beads to yield the final library. The library preparation was performed using the NEBNext® Ultra™ RNA Library Prep Kit for Illumina®. Following library construction, an initial quantification was performed using the Qubit 2.0 Fluorometer. The library was diluted to 1.5 ng/μl, after which the insert size was assessed using the Agilent 2100 Bioanalyzer. Once the insert size met expectations, qRT-PCR was employed to accurately quantify the effective library concentration (above 2 nM), thereby ensuring library quality.