title: GSM8439471: g_MGKC_3; Gallus gallus; RNA-Seq
library_construction_protocol: Total RNA was extracted using TRIzol reagent (ET101-01-V2, Beijing TransGen Biotech Co., Ltd, Beijing, China). The RNA concentration and integrity were detected using a Fragment Analyzer (Agilent, California, USA). RNA samples that complied with the criteria of having a concentration of more than 40 ng/μL, an integrity value greater than 7.0, and a total content of 1 μg or more were chosen for further experimentation. Oligo dT beads were employed to enrich mRNA carrying a poly(A) tail. Subsequently, the RNA was fragmented, and first-strand cDNA production was initiated through random N6-primed reverse transcription. This was followed by the synthesis of second-strand cDNA, where dUTP was utilized instead of dTTP. The resulting cDNA then underwent end-repair and subsequent 3' adenylation. Afterward, adaptors were attached to the termini of these 3' adenylated cDNA segments. Before initiating PCR amplification, the strand marked with dUTP was selectively degraded using Uracil-DNA-Glycosylase (UDG). The remaining strand was then amplified to create a cDNA library suitable for sequencing. To enrich the purified cDNA template, multiple rounds of PCR amplification were carried out using PCR primers. Heat was used to denature the PCR product, and subsequently, the single-stranded DNA was circularized with the aid of a splint oligo and DNA ligase. Ultimately, DNA nanoball synthesis and sequencing were executed on the DNBSEQ platform in paired-end 150 sequencing mode.