protein G Dynabeads (ThermoFisher Scientific #100009D) coated with anti-SFMBT2 generated inhouse (Miri K et al., 2013)
Sequenced DNA Library
library_strategy
ChIP-Seq
library_source
GENOMIC
library_selection
ChIP
library_construction_protocol
[library prep] NEB Ultra DNA Cell lysis and genomic fragmentation methods varied according to the intended fragmentation method -- sonication or micrococcal nuclease digestion. Briefly, cells were sonicated to an average size of 75 bp to 400 bp using a total processing time of 5 mins with an amplitude of 40, pulse duration of 10 s, and cooling duration of 30 s. Samples fragmented by micrococcal nuclease digestion were on average 175 bp in size. DNA crosslinked with endogenous SFMBT2 was immunoprecipitated using magnetic Protein G beads coated with anti-SFMBT2 antibody produced in house. FLAG-SFMBT2 complexes were immunoprecipitated with magnetic FLAG-conjugated beads. Immunoprecipitation was performed at 4C for 2 hrs and subsequently washed 5X with cold RIPA buffer and once with cold TE supplemented with 50 mM NaCl. Complexes were decrosslinked in 1% SDS and 0.1 M NaHCO3 overnight at 65C. DNA was subsequently extracted through phenol-chloroform extraction and purified by ethanol-based precipitation. DNA was reconstituted in water and quantified using PicoGreen. Sonicated endogenous SFMBT2 ChIP and input DNA libraries were generated using the Illumina TruSeq protocol. Sonicated FLAG-SFMBT2 and all micrococcal nuclease-digested ChIP and input DNA libraries were prepared using the NEB Ultra DNA library preparation protocol.