ChIP-seq IP of SigE Synechocystis sp. PCC6803 rep.1
The overall method was according to the previous study (Hanaoka and Tanaka 2008) with the following modification. Cells equivalent to 120mL culture (8 tubes) were used for SigE-FLAG ChIP-seq experiment, 60mL for SigA-FLAG ChIP-seq, and 30mL for ChIP-qPCR experiment. Each 15mL cultures were put into 2mL tubes and 100L of acid-washed glass beads (G4415) and 50 L of lysis buffer (50mM HEPES-KOH pH7.5, 140mM NaCl, 1mM EDTA, 1% Triton-X100, 0.1% deoxycholate) supplemented with Complete Mini EDTA-free (Roche) and 1mM PMSF. Cells were beads-beaten with shakemaster NEO by five times repeat of 1min 1500rpm and 1min interval on ice. After beating, 500L of lysis buffer was added per 2 tubes, and cell lysate was collected. In order to shear DNA into 200bp, cell lysate was subjected to sonication (24 cycles of 10sec 20% amplitude and 50sec interval on ice by VC-750, EYELA, Tokyo, Japan). After removing debris by centrifuge (18,000 xg 5min), protein concentration was measured by Pierce BCA Protein Assay Kit, and the lysate was diluted to 4mg protein /mL protein by lysis buffer. As the input of IP, 20L of lysate was diluted with 80L of elution buffer (10mM Tris-HCl pH8.0 100mM NaCl 0.1%SDS). Before the immunoprecipitation, Anti-FLAG M2 Affinity Gel (A2220 Sigma Aldrich) was equilibrated with PBS containing 3% BSA. For immunoprecipitation, 20L bed volumes of Anti-FLAG M2 Affinity Gel were added to cell lysate for ChIP-seq assay and rotated for 2hours at 4C. Beads were collected by centrifuge (800 xg 1min), and washed twice with lysis buffer, once with high-salt lysis buffer (50mM HEPES-KOH pH7.5, 500mM NaCl, 1mM EDTA, 1% Triton-X100, 0.1% deoxycholate), twice with ChIP Wash Buffer (50mM Tris pH8.0 250mM LiCl 0.5% NP-40 0.5% Deoxycholate, two-fold dilution of SC-45002 Santa Cruz), and once with TE buffer (10mM Tris-HCl pH7.5 1mM EDTA). Then beads bound DNA-protein complex was eluted with elution buffer for 30min at 60C. The eluted solution and input were treated with 1mg/mL (final concentration) of RNAse A (Nippon Gene, Tokyo Japan) for 30minutes at 37˚C, then 0.5mg/mL proteinase K (Kanto Chemical, Tokyo Japan) were added and incubated at 65C for overnight. Then the DNA was extracted with FastGene Gel/PCR Extraction Kit (NIPPON Genetics, Tokyo, Japan). For Library preparation and Next-generation sequencing, input and immunoprecipitated DNA were prepared into multiplexed libraries with ThruPLEX DNA-Seq Kit (Takara bio, Shiga, Japan) and DNA Unique Dual Index Kit (Takara). Multiplexed libraries were paired-end sequenced with HiSeqX.