GSM8265723: Monocytes, IgG, RIP; Homo sapiens; RIP-Seq
Monocytes were lysed and RNA immunoprecipitation (RIP) was performed using the Magna RIP® RNA Binding Protein Immunoprecipitation Kit (Sigma-Aldrich, #17-700) according to manufacturer's instructions. The following antibodies were used: normal rabbit IgG (Nordic Immunology, #RIgG), rabbit anti-S100A9 (0.328 µg; CellSignalling, #72590), rabbit anti-S100A8 (2 µg; ThermoFisher, #MA5-36007). RNA immunoprecipitation was validated by immunoblotting. RNA sequencing library was prepared using the NEBNext Ultra II RNA Library Prep Kit for Illumina (New England Biolabs) according to manufacturer's instructions. The RNA was fragmented at 94 °C for 15 minutes. First strand and second strand cDNA were subsequently synthesized. cDNA fragments were end repaired and adenylated at 3'ends, and universal adapters were ligated to cDNA fragments, followed by index addition and library enrichment by PCR with limited cycles. Sequencing library was validated on the Agilent TapeStation (Agilent Technologies), and quantified by using Qubit 2.0 Fluorometer (ThermoFisher Scientific). Sequencing libraries were multiplexed and clustered onto a flow cell on the Illumina NovaSeq instrument according to manufacturer's instructions. Samples were sequenced using a 2x150bp Paired End (PE) configuration. Image analysis and base calling were conducted by the NovaSeq Control Software (NCS).