GSM9724862: Liver, LPS, day 11, ZT21, rep3; Mus musculus; RNA-Seq
Total RNA was extracted from the samples for mRNA sequencing. The report states that mRNA-seq library preparation required 1 ug total RNA. RNA integrity was assessed by agarose gel electrophoresis with a 28S:18S ratio >= 1.5, and total RNA quantity was measured by Qubit with >= 500 ng required for library preparation. mRNA was enriched from total RNA using Oligo(dT)-coupled magnetic beads. Enriched mRNA was fragmented into ~200 bp fragments. First-strand cDNA was synthesized using random primers, and strand-specific second-strand cDNA synthesis was performed with dUTP in place of dTTP. Double-stranded cDNA was purified with AMPure XP beads, end repaired, A-tailed, and ligated to sequencing adapters. The second cDNA strand was digested with USER enzyme, PCR amplified for approximately 15 cycles, and the final library was quality checked and quantified. Sequencing was performed on an Illumina NovaSeq 6000 platform in paired-end 2 x 150 bp mode.