title: GSM8370847: kd-1; Sus scrofa; RNA-Seq
library_construction_protocol: Aspirate the culture medium from the culture dish and wash once with 1 mL of PBS. Aspirate the PBS (removing as much as possible) and add 1 mL of TRIzol. Incubate for 5 minutes, gently pipette against the bottom of the dish, then aspirate the TRIzol and transfer it to a 1.5 mL centrifuge tube. Allow it to stand at room temperature for 5 minutes. Add 250 µL of chloroform, vortex for 30 seconds. Allow it to stand at room temperature for 5 minutes. Centrifuge at 12,000 rpm for 5 minutes; carefully transfer the aqueous phase to another 1.5 mL centrifuge tube using a pipette. Add 500 µL of isopropanol to the aqueous phase, mix gently, and incubate at 4°C overnight. Centrifuge at maximum speed (12,000 rpm) for 20 minutes. Discard the isopropanol, wash the pellet with 1 mL of 75% ethanol. Centrifuge at 12,000 rpm for 5 minutes. Discard the ethanol and air-dry the pellet. Dissolve the RNA pellet in approximately 30 µL of DEPC-treated water and store at -80°C. 1Two biological RNA samples from two independent experiments were sent to Frasergen (Wuhan, China) for mRNA sequencing library construction.Briefly, these libraries were sequenced using the MGI high-throughput sequencing platform. Raw reads were filtered using SOAPnuke (v2.1.0) software to obtain clean reads, which were then aligned to the pig genome (Sscrofa 11.1) using HISAT2 (v2.2.1). The quality-controlled second-generation sequences were subsequently mapped to the reference transcriptome sequences using bowtie2 (v2.3.5) software. RSEM (v1.3.3) was used to count the number of reads mapped to each transcript in each sample and convert them to FPKM (Fragments Per Kilobase of transcript per Million mapped reads), thereby obtaining the expression levels of genes and transcripts. Finally, the DESeq2 algorithm was employed with a moderate cutoff value (Pvalue < 0.05) to filter differentially expressed genes (DEGs).