title: GSM8603286: Blood, CAD vaccine immunized, pre-immunization (Day 0)，rep1; Mus musculus; RNA-Seq
library_construction_protocol: Total RNA was isolated from samples using the Trizol Reagent (Invitrogen Life Technologies). Following extraction, the concentration, quality, and integrity of the RNA were assessed using a NanoDrop spectrophotometer (Thermo Scientific). Three micrograms of RNA were then used as input material for the RNA sample preparations. mRNA was purified from the total RNA using poly-T oligo-attached magnetic beads to selectively isolate polyA RNA for subsequent sequencing. Sequencing libraries were constructed by purifying mRNA from total RNA using poly-T oligo-attached magnetic beads. The RNA was fragmented with divalent cations in an Illumina proprietary buffer. First strand cDNA was synthesized using random oligonucleotides and Super Script II, followed by second strand cDNA synthesis with DNA Polymerase I and RNase H. Overhangs were converted to blunt ends, and the enzymes were removed. After adenylation of the 3' ends, Illumina PE adapter oligonucleotides were ligated. cDNA fragments of 400-500 bp were purified using the AMPure XP system. DNA fragments with ligated adapters were enriched using Illumina PCR Primer Cocktail in a 15-cycle PCR reaction. The products were purified and quantified using the Agilent high sensitivity DNA assay on a Bioanalyzer 2100 system.