mRNA-Seq of rat: brain
Sample Lysis: Grind tissue samples (30 mg) with 600 L Buffer RLT Plus. Homogenize and incubate for 5 minutes. Centrifuge at 12,000 g for 3 minutes and transfer the supernatant to an AllPrep DNA spin column. Purify RNA by centrifugation.DNA Purification: Add 500 L Buffer AW1 to the column and centrifuge. Repeat with Buffer AW2, followed by Buffer EB to elute DNA.RNA Purification: Mix collected RNA with 70% ethanol. Transfer to RNeasy spin column, centrifuge, and repeat. Wash with Buffer RW1 and Buffer RPE, then elute RNA with RNase-free water.Sample Testing: Perform quality checks on RNA samples. Isolate mRNA using oligo(dT) beads, fragment, and synthesize cDNA. Amplify and validate libraries. Circularize single-stranded cDNA and generate DNA nanoballs (DNBs) for sequencing.Data Analysis: Filter raw sequencing data using SOAPnuke. Assemble clean reads with Trinity, assess quality, align to reference sequences with Bowtie2, and calculate expression levels with RSEM.