GSM8283722: KP-1 Cells, Carfilzomib Treatment for 48 hours, rep1; Mus musculus; RNA-Seq
RNA was harvested using TRNzol Universal Total RNA Extraction Reagent Kit (DP424, TIANGEN). 1 μg of total RNA was used for the construction of sequencing libraries. mRNA sequencing is based on the Illumina platform, which sequences all transcribed mRNA. The sequencing experiment utilizes the Illumina TruseqTM RNA sample prep Kit method for library construction, with the following specific operational steps: 1. Total RNA is extracted from the sample, and its concentration and purity are assessed using Nanodrop2000. RNA integrity is checked via agarose gel electrophoresis, and the RIN value is determined using Agilent2100. For a single library preparation, RNA totaling 1 μg is required, with a concentration ≥50 ng/μL and an OD260/280 ratio between 1.8 and 2.2. 2. Utilizing magnetic beads with Oligo(dT) to pair with polyA, mRNA is separated from total RNA for transcriptome analysis. 3. Fragmentation buffer is added, and under suitable conditions, mRNA is randomly fragmented into approximately 300 bp fragments. Subsequently, using reverse transcriptase and random primers, a single-stranded cDNA is synthesized from the mRNA template, followed by second-strand synthesis, forming a stable double-stranded structure. 4. The double-stranded cDNA structure has sticky ends, which are repaired to blunt ends by adding End Repair Mix. Then, an A base is added to the 3' end to prepare for adapter ligation. 5. The adapter-ligated products are purified and fragmented, followed by PCR amplification using the selected fragments to obtain the final library. 6. Quantification is performed using the QuantiFluor® dsDNA System, and the samples are mixed according to data proportions. Bridge PCR amplification is conducted on the cBot to generate clusters, followed by sequencing using the Illumina platform.