title: GSM5640212: 4_NR4A1; Homo sapiens; ChIP-Seq
library_construction_protocol: After culture, cDCs were washed with PBS and crosslinked using the truChIP® Ultra-Low Chromatin Shearing Kit (Covaris), according to the manufacturer's instructions. Chromatin shearing was performed using AFA Fiber Pre-Slit Snap-Cap microTUBEs (Covaris) by sonication (peak incident power: 105, duty factor: 2%, cycles per burst: 200, treatment time: 12 minutes) on the Covaris S220 focused ultrasonicator (Woburn, MA, USA). After shearing, the chromatin was transferred into pre-chilled microcentrifuge tubes and centrifuged at 10.000 x g, 4˚C for 5 minutes to pellet insoluble material, and the chromatin was stored at -20˚C for downstream processing. Chromatin immunoprecipitation was performed with 3µg anti-NR4A1 (NB100-56745, Novusbio), anti-NR4A2 (NB110-40415, Novusbio) or anti-NR4A3 (NLS2341, Novusbio) antibodies, using the low cell ChIP-seq kit (Active Motif), according to the manufacturer's instructions. For all conditions, 10% of the input chromatin was removed prior to addition of the antibodies and used to normalize the amount of immunoprecipitated DNA (input control). Following immunoprecipitation, ChIP DNA was de-crosslinked in the presence of NaCl and Proteinase K (Active Motif) in a thermocycler at 65°C overnight, and DNA was extracted by penol:chloroform:isoamylalcohol precipitation and dissolved in Low-EDTA TE buffer (Active Motif). ChIP-seq libraries were generated by GenomeScan (Leiden, the Netherlands) with the NEBNext® Ultra II DNA Library Prep kit (Illumina), and were sequenced using Illumina NovaSeq 6000 generating ~20 million 150bp paired ended reads for each sample.