title: GSM7245943: 10870-CovR - output - 3 - biol rep 1; Streptococcus pyogenes; ChIP-Seq
library_construction_protocol: Three biological replicates of strains MGAS10870, M3-CovS-T284A, and 10870-ΔcovR (control), respectively, were chromatin immunoprecipitated using polyclonal antibody directed against the N-terminal part of CovR. Briefly, GAS strains were grown in 40 ml THY medium to mid-exponential phase (OD~0.45), proteins were cross-linked to DNA with 1% formaldehyde, cells harvested by centrifugation, and flash-frozen and stored at -80° C. The fixed cell pellets were resuspended in 1ml ice cold lysis buffer, and the lysates were sonicated for 15 cycles (30s on/30s off) at 4° C in 1.5 ml Bioruptor tubes (Diagenode) in a Diagenode Bioruptor Plus machine set at high power, to shear DNA to fragments of 200 and 400 bp length.  The cleared supernatant was collected to use for chromatin-immunoprecipitation (950μl) or input DNA (50μl), respectively. CovR-bound DNA fragments were immunoprecipitated over night at 4°C using Dynabeads protein G (Invitrogen) pre-coated with anti-CovRND antibody.  After several washing steps, the complex was eluted with 50μl elution buffer to obtain the ChIP DNA (output) samples. Proteins and RNA in both input and ChIP samples were degraded using RNaseA and proteinase K, crosslinking was reversed by incubation at 65° C overnight, and the DNA was purified using SPRI beads (AMPure XP, Beckman Coulter) on a magnetic stand. The DNA concentration was determined on a Qubit machine 4.0 (Invitrogen) following the Qubit manual for high-sensitive DNA, and DNA fragment size distribution was assessed using Agilent D1000 Screen Tape on an Agilent 2200 TapeStation system. ChIP sequencing was performed in the Advanced Technology Genomics Core (ATGC) Facility at MD Anderson Cancer Center] with Illumina compatible indexed libraries prepared from 2-10ng of sheared ChIP or input DNA.   Equimolar quantities of the indexed libraries were multiplexed with 8 libraries per pool and sequenced on an Illumina NextSeq500 sequencer using the high output 75nt single read flow cell format.