title: Illumina HiSeq 2500 sequencing; Systematic interaction profiling of human bromodomain-containing proteins reveals complex modes of binder recruitment for BET proteins
design: Systematic interaction profiling of human bromodomain-containing proteins reveals complex modes of binder recruitment for BET proteins
library_construction_protocol: Flp-In T-REx U-2 OS cells stably expressing GFP tagged BRD3 WT or a N116A N391A construct (bd(1+2)) were grown in 2 x 15 cm plates to ~75% confluence and induced with 1 µg/mL tetracycline for 48 hours. Cells were then washed with PBS and crosslink with 1% formaldehyde in PBS for 15 minutes at room temperature before quenching the reaction with 125 mM glycine in PBS for 5 minutes at room temperature. Cells were then pelleted and frozen at -80°C until ChIP was performed.  Chromatin immunoprecipitation (ChIP) was performed as described previously (REF, PMID:19275939) using 7 µg of anti-GFP (ab290, Abcam) antibody.  220 ng of input control DNA extracted from sonicated cell lysates of each sample were processed in parallel.  ChIP DNA was prepared for Illumina sequencing by blunt-end repair, dA-tailing, and ligation of Illumina adaptors using a NEBNext DNA library preparation kit (New England Biolabs, catalogue #E6040L). Total ChIP DNA (approximately 200-500ng) and 220ng of DNA input (WCE) was end repaired for 30 minutes at room temperature, and then purified using column purification with either DNA Clean and Concentrator (Zymogen, catalogue #D4014) or PCR purification columns (Qiagen, catalogue #28106) as recommended by manufacturer's protocol. Blunt-end repaired DNA was dA-tailed for 40 minutes at 37° C, then column purified. dA-tailed DNA was ligated to Illumina adaptors (final concentration 6.67 nM) that have a T- overhang. USER enzyme was used to cleave the uracil hairpin of the Illumina adaptor, and adaptor-ligated DNA was column purified. The library was PCR amplified for 16-18 cycles using a universal primer and a barcoded primer (New England Biolabs, catalogue #E7335L). PCR-amplified DNA was purified using PCR column purification and eluted in 20 uL of elution buffer for preparation of gel extraction, or 30 uL of TE for preparation of Pippin Prep size selection. Library samples were size selected from 200-350bp using a 2% agarose dye-free automated size selection cassette from Pippin Prep (Sage Sciences, catalogue #CDF2010).
attributes.tag: Experimental Factor: immunoprecipitate
attributes.value: anti-GFP (ab290)
attributes.tag: Experimental Factor: genotype
attributes.value: mutant BRD3