5300 fragment analyzer ngs fragment kit

Cells were treated with or without IFNβ for 48 hrs, harvested, and RIP conducted using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore) following the manufacturer’s instructions. Briefly, cell pellets were lysed in RIP lysis buffer, followed by incubation with RIP Buffer containing magnetic beads conjugated with Z-RNA or isotype control antibody at 4°C overnight. Samples were then incubated with proteinase K and immunoprecipitated RNAs were recovered by phenol:chloroform:isoamyl alcohol purification. RNA was quantified using the Quant-iT RiboGreen RNA assay (Thermo Fisher Scientific) and assessed for quality with the 2100 Bioanalyzer RNA 6000 Nano assay (Agilent) or 4200 TapeStation High Sensitivity RNA ScreenTape assay (Agilent) prior to library generation. Libraries were prepared from total RNA with the TruSeq Stranded Total RNA Library Prep Kit according to the manufacturer’s instructions (Illumina, PN 20020599). Libraries were analyzed for insert size distribution using the 2100 BioAnalyzer High Sensitivity kit (Agilent), 4200 TapeStation D1000 ScreenTape assay (Agilent), or 5300 Fragment Analyzer NGS fragment kit (Agilent). Libraries were quantified using the Quant-iT PicoGreen ds DNA assay (Thermo Fisher Scientific) or by low pass sequencing with a MiSeq nano kit (Illumina). Paired-end 150 cycle sequencing was performed on a NovaSeq 6000 (Illumina).