GRO-Seq analysis of Wt mouse BMDM cells differentiated in the presence of MCSF (1hIL4)
Macrophages were treated and nuclei isolation was performed using buffer containing 10mM Tris-HCl, pH 7.4; 2mM MgCl2; 3mM CaCl2; 0.5% NP40; 10% Glycerol; 1mM DTT supplemented with protease inhibitors (prior to use) and RNase inhibitor (SUPERase-In, prior to use). Nuclear Run-On (NRO) reactions were performed in the presence of Br-UTP in duplicates containing ~10x106 nuclei and pooled for nascent RNA enrichment, using BrdU antibody coated agarose beads. Beads were washed and NRO RNA was eluted and quantified by Qubit fluorometer. Approximately 100ng NRO RNA was used for end-repair with T4 PNK (3_-end de-phosphorylation) and Cap-Clip Acid Pyrophosphatase (de-capping). Libraries were prepared with NEBNext Small RNA Library Prep set for Illumina and sequenced on HiSeq 2500.