GSM6503009: 3C-seq of E. coli wt cells in stationary phase 37°C -Replicate 2; Escherichia coli str. K-12 substr. MG1655; OTHER
Fresh formaldehyde solution with a final concentration of 7% (1%, 3%, 5% and 7% were used in the comparison experiment of formaldehyde) was added to 1mL of E. coli culture medium, and the chromosomes were fixed by incubating at room temperature for 30 min and then incubating at 4℃ for 30 min. The excess formaldehyde was quenched by adding 2.5 M glycine solution and incubating at 4 ° C for 30 min. Centrifugation was performed at 3500 g for 10 min at 4 ° C and the supernatant was removed. The precipitate can be stored in refrigerator at -80℃ or digested directly. 10 μL protease inhibitors and 200 μL Ready-Lyse were added to each tube of precipitate, and the precipitate was resuspended and incubated for 30 min at room temperature. Centrifugation was performed at 3500 g for 10 min at 4 ° C and the supernatant was removed. The precipitate obtained can be directly used for the next enzyme digestion, or it can be quick-frozen with liquid nitrogen and stored in the refrigerator at -80℃ for about a week for subsequent experiments. The fixed precipitated product was resuspended with 100 μL of 1×TE Buffer, and 5 μL of 10%SDS was added and incubated for 20 min at room temperature to obtain the lysate solution of bacteria. If it is to use frozen precipitation products need to be placed on ice in advance to thaw. Each 100 μL lysate solution was mixed with 50 μL 20% TritonX-100, 100 μL Cut Smart Buffer, 710 μL ultra-pure water and 40 μL limiting enzyme HpaII (or Hpy4CHIV). When configuring the digestion buffer, it is best to perform the operation on ice and limit the enzyme to the last addition. The digested buffer system was incubated at 37℃ for 6 h (treated for 3 h, 5 h, and 6 h respectively in the enzyme digestion time experiment) to fully fragment the DNA, and then 50 μL 10% SDS was added to quench the excess restriction enzymes. Before joining, the digested solution was cooled on ice for a period of time, ensuring that the temperature did not exceed 16 °C. Because the connection system requires a lot of dilution, it is best to also cool ultrapure water on ice to below 16 ° C. To each 1 mL of enzymatic solution, 100 μl BSA, 1 mL 20% TritonX-100, 2 mL T4 Ligation Buffer, 1.6 mL ultrapure water, and 10 μl T4 DNA ligase were mixed thoroughly. At this stage, after all other solutions have been added, let cool on ice for a few minutes before adding the ligase. The connecting system was placed in a water bath in an incubator at 16℃ for 4 hours and then 200 μL 0.5 M EDTA was added and mixed. Proteinase K was added to the mixture and the final concentration of 100 ug/mL was incubated overnight at 58 ° C to digest the protein in the solution. The obtained mixed solution was thoroughly mixed with an equal volume of phenol chloroform isoamyl alcohol DNA extract and centrifuged at 12000 g for 10 min. The top layer of nucleic acid solution was transferred to a new EP tube and 1/10 volume of 3 M sodium acetate pH = 5.2, an appropriate amount of Glycogen and twice the ice-cold absolute ethanol were added for mixing. The supernatant was removed after centrifugation at 12000 g at 4 ° C for 15 min. The precipitate was redissolved with 100 μL of 1×TE Buffer and RNaseA was added and incubated for 30 min at 37 ° C for RNA degradation. After the DNA samples passed the test, the Covaris ultrasonic crushing instrument was used to randomly interrupt the library, and then the whole library preparation was completed through the steps of end repair, adding A-tail, adding sequencing adaptor, purification, PCR amplification and so on. After the construction of the library, Qubit 2.0 was used for preliminary quantification and dilution of the library, and then Agilent 2100 was used to detect the inserted fragments of the library. After the size of the inserted fragments met the expectation, Q-PCR method was used to accurately quantify the effective concentration of the library to ensure the quality of the library. 3C-seq