title: GSM5097997: D39 ΔbgaA:: PspxB-metR-3×flag no methionine IP 2; Streptococcus pneumoniae D39; ChIP-Seq
library_construction_protocol: The culture was grown to OD620 0.2 before being washed being mixed with 1% formaldehyde in the fixation buffer at a volume ratio of 1:10 to crosslink interacting bacterial protein-DNA molecules. The mixture was gently rotated for 30 min at room temperature, quenched 1:3 (v/v) with 1 M Tris, pH 8.0, for 10 min, pelleted by centrifugation at 4°C, washed twice in ice-cold PBS by centrifugation, and snap-frozen in liquid nitrogen.  The cell pellets were then resuspended in 2 ml ice-cold lysis buffer in a 5-ml round-bottom tube, and sonicated on ice for 55 X 5 seconds with an interval of 20 seconds to break down genomic DNA using Sonics Vibracell VC750 with 30 % amplitude.  After centrifugation at 4°C, the supernatants were divided into the 200 μl input control and 800 μl immunoprecipitation (IP) sample.  The IP sample was mixed with the lysis buffer with 10 μg anti-FLAG antibody (Sigma) on a rotating wheel for 8 h at 4 °C, and subsequently with protein G-coupled Dynabeads (Invitrogen) for 10 h at 4 °C.  Beads were isolated on a magnet, sequentially washed with the lysis and wash buffers by centrifugation, resuspended in 520 µl TES buffer, and incubated at 65 °C for 4 h to de-crosslink and elute DNA fragments from beads.  The input sample was supplemented with 300 µl TES and 20 µl 10 % SDS and incubated at 65 °C for 4 h.  Both the IP and input samples were treated with 12.5 μl Proteinase K (20 mg/ml in TE) before DNA was purified by sequential treatment with phenol, chloroform, and ethanol.  DNA pellets were dissolved in 100 μl TE (pH 8.0) for 15 min at 65 °C.  The DNA samples were verified by DNA gel electrophoresis and used to construct libraries. The purified DNA was used for ChIP-seq library preparation. The library was constructed by Novogene Corporation (Beijing, China). Subsequently, pair-end sequencing of sample was performed on Illumina platform (Illumina, CA, USA). Library quality was assessed on the Agilent Bioanalyzer 2100 system.