title: GSM9523109: E. coli Hha deletion, Sfx deletion R6K, Psfx-sfx-FLAG, rep 2 (ChIP); Escherichia coli K-12; ChIP-Seq
library_construction_protocol: ChIP-seq samples of FLAG-tagged Sfx and H-NS. The crosslinked cell pellets were resuspended in 1x IP buffer (200 mM Tris, pH 8.0, 600 mM NaCl, 4% Triton X-100, 2x ProBlock Gold 2D Protease Inhibitor Cocktail). The resuspended cells were subjected to sonication to yield DNA fragments ranging from 100 to 500 bp. The cells were kept in an ice-water bath throughout sonication. After sonication, 0.1 volume of cell lysate, which is used as the input sample, was kept at 4 ℃ until reverse-crosslinking. The remaining cell lysate was mixed with MonoRab anti-DYKDDDDK magnetic beads (GenScript). After an overnight incubation at 4 ℃, the magnetic beads were subjected to sequential washes, with 1 ml used per wash: 1) 1x Wash buffer A (100 mM Tris-HCl, pH 8.0, 250 mM LiCl, 2% Triton X-100, 1 mM EDTA); 2) 1x Wash buffer B (100 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1% Triton X-100, 0.1% sodium deoxycholate, 1 mM EDTA); 3) 1x Wash buffer C (10 mM Tris-HCl, pH 8.0, 500 mM NaCl, 1% Triton X-100, 1 mM EDTA); 4) 1x TE (10 mM Tris-HCl, pH 8.0, 1 mM EDTA). The antigens were eluted in 1 ml of elution buffer (50 mM Tris, pH 8.0; 10 mM EDTA; 1% SDS, 5 U/ml proteinase K (NEB, cat# P8107S)) by a one-hour incubation at 65 ℃, with vigorous vortexing every 10 minutes. The input samples were diluted 10 times with elution buffer and subjected to the same treatment as the ChIP samples from now on. The samples were incubated at 65 ℃ overnight to reverse the crosslinking. To digest the RNA, 100 μg/ml of RNase A (ThermoFisher) was added for 2 h incubation at 37 ℃, followed by 2 h incubation at 50 ℃ with 5 U/ml of proteinase K (NEB). Finally, DNA was recovered using QIAquick PCR Purification Kit (QIAGEN). ChIP-seq samples of RNAP. The protocol is largely the same as FLAG-tagged ChIP-seq samples except for the incubation with antibodies. The samples were first incubated with Protein A/G MagBeads (GenScript) for 3 hours at 4℃, and then the protein A/G MagBeads were removed. Anti-E. coli RNA polymerase β antibody was added to the samples for an overnight incubation. The next day, Protein A/G MagBeads were added again. After 3 hours of incubation, the magnetic beads were washed as above. RNA ChIP-seq samples. The protocol is largely the same as FLAG-tagged ChIP-seq sample preparation with the following changes. After reverse crosslinking, the samples were treated with Turbo DNase (ThermoFisher) and then the RNA was purified by Monarch Total RNA Miniprep Kit (NEB). DNA and RNA libraries were prepared using Illumina DNA Prep, (M) Tagmentation kit and Illumina Stranded mRNA Prep kit, respectively, following the manufacturer's protocols.