RNA-Seq of mus musculus: spinal cord
For each sample, 200 ng of qualified RNA was used for library preparation. mRNA was enriched from total RNA using oligo(dT)-attached magnetic beads. The enriched mRNA was fragmented with a fragmentation buffer and reverse-transcribed using random N6 primers to synthesize double-stranded cDNA. The cDNA was end-repaired, 5'-phosphorylated, and had a protruding 'A' added to the 3' end. Bubble-shaped adapters with a protruding 'T' at the 3' end were ligated to the blunt-ended DNA. The ligated products were amplified using specific primers via PCR. PCR products were denatured to single strands, and single-stranded circular DNA libraries were generated using a bridged primer. The libraries were subjected to QC before sequencing. DNA nanoballs (DNBs) were prepared through phi29 amplification, generating more than 300 copies of each molecule. These DNBs were loaded onto a patterned nanoarray, and paired-end 150-base reads were sequenced using the MGI T7 platform via synthesis.