Cell lines (S2, Kc167...) are grown as described by the DGRC and the Cherbas Lab. After expanding the cell line, cells are transfered in a plate. Once confluent, 1.8% formaldehyde is added to the cell culture. Cells are harvested in presence of the formaldehyde with the help of a cell scrapper. After 15 minutes of incubation at room temperature, the cross-linking reaction is quenched with glycine for 5 minutes. Cells pellet are then washed 3 times with lysis buffer. Regular chromatin extraction before sonication is then performed. Formaldehyde Cross-linking of Chromatin from any Drosophila tissue (Embryos, Larvae, Pupae, Adult flies).1. Homogenize the material (about 150-200 mg of embryos, pupae, adµlts, larvae, sufficient for four-five independent immuno-precipitations) in 5 ml of buffer A1 + 1.8% formaldehyde (290 µl of 37% solution) at room temperature using first a Potter homogenizer and then a Douncer with type A pestle (3 strokes). Wait 15? (total time starting from beginning of homogenization). Add glycin solution to 225 mM (540 µl of 2.5 M solution for 6 ml of cross-linked mixture), mix and incubate 5?. Put on ice. Keep on ice unless specified.2. Transfer the homogenate to a 15 ml tube. Centrifuge 5 min, at 4000 g at 4°C. Discard supernatant. Add 3 ml of buffer A1, resuspend pellet and spin down the same way. Repeat washing step 3 times.3. Wash once in 3 ml of lysis buffer without SDS. Spin down, 5 min at 4000g.4. Resuspend cross-linked material in 0.5 ml of lysis buffer and add SDS to 0.1% (2.5 µl) and N-lauroylsarcosine to 0.5% (12.5 µl). Incubate 10 min at 4°C in a rotating wheel.5. Sonicate in Polypropylene 15ml Falcon tubes according to .Diagenode instructions. The settings are: High power, 15 minutes, 30 seconds off, 30 seconds on. The water bath shoµld be kept cold either in a cold room or by a cold water pump.6. Rotate 10'. Transfer to eppendorf tubes and centrifuge 5', room temperature at maximum speed. Transfer supernatant to the new tube. Add another 0.5 ml of lysis buffer to the pellet, rotate for 10 min. Repeat centrifugation and combine supernatants. Centrifuge the combined supernatant 2x10 min at max speed. The supernatant from this stage will be referred as chromatin extract. Chromatin can be stored at ?80°C for several months at this stage. Add sodium azide to 0.02% for storage.7. [Optional : Purification of chromatin. Put chromatin extract in Centricon YM-100 column (4212), centrifuge 3x40' (or more) at 1000 g while adding lysis buffer. At least 3 vol. of lysis buffer should pass through column. Bring the final volume of chromatin extract to 1 ml by lysis buffer + protease inhibitors.] Solutions and MaterialsLysis buffer: (see above)5mg/ml RNaseA (DNase free)PAS suspension: 100 mg of CL-4B (Amersham, 17-0780-01) PAS shoµld be resuspended in 1 ml of lysis buffer + protease inhibitors + 0.1 mg/ml BSA for 50 %v suspension. Wash in lysis buffer 2-3 times, overall equilibration time 1h. Store up to one week at 4 C.TE: 10 mM Tris-HCl pH 8.0, 1 mM EDTAElution buffer1: 10 mM EDTA, 1% SDS, 50 mM Tris-Cl pH 8.Elution buffer2: TE+0.67% SDS.LiCL 4M? Protocol 1. To an amount of chromatin corresponding to 150 mg of biological material, suspended in a final volume of chromatin extract of 1 ml (in lysis buffer + protease inhibitors), add 100 µl of PAS suspension for preincubation. Incubate several hours or overnight at 4°C, then remove PAS. Crosslinked chromatin at this stage can be stored several days at 4°C or frozen at ?70°C.2. [Optional : Check for amount of DNA. From the 1 ml solution above, take a 100 µl aliquot, Add proteinase K up to 100 ug/ml and SDS to 1%, incubate 6h at 60°C, then 20 min at 70°C, add RNAse to 50 ug/ml and incubate additional 2h at 37°C. Phenol-chlorophorm extract, ethanol precipitate. Run on an agarose gel to check amount and size of DNA.]3. Separate chromatin sample into 4x250 µl aliquots (one aliquot is enough for one IP but the amount may be decreased further). Immunoprecipitate chromatin by adding the antibody (Ab) of interest (amount of Ab shoµld be determined empirically, usually the same concentration than for a successfµl IF experiement). Keep a control sample without Ab IP, named "Mock".4. Incubate 4 h at 4°C on a head to head rotating wheel, then add 50 µl of PAS suspension and incubate 4 h or overnight. Spin down PAS and proceed to washes.5. Wash PAS 4x with lysis buffer, followed by 2x with TE (without protease inhibitors). Each wash is for 5 min at 4°C, using 1 ml of solution.6. Elution of precipitated material. Spin down PAS. Add 100 µl of elution buffer, mix and incubate 10 min at 65°C. Spin down PAS and transfer supernatant to new tube. Add 150 µl of elution buffer 2 to PAS, mix, centrifuge at fµll speed and transfer eluate to a tube together with the eluate from the first centrifugation. The combined material is the "chromatin precipitate" (approx. 250 µl).7. Incubate precipitate 6 h (or overnight) at 65 C to reverse cross-links. Add 250 µl of Proteinase K solution, incubate at 50°C 2-3 h.8. Add 55 µl of 4M LiCl and 500 µl of phenol-chlorophorm. Mix and microfuge at fµll speed at RT. Transfer aqueous phase to a new tube and precipitate with 1 ml of 100% ethanol. Wash with 750 µl of 70% ethanol. Spin down and dry precipitate.9. Dissolve in 25 µl of water. This is the chromatin immunoprecipitate or ?Native ChIP? sample. DNA fragments recovered following chromatin IP prepared for Illumina sequencing using the Epicentre Nextera DNA Sample Prep Kit (Cat. # GA0911). After a final gel purification step the DNA is loaded into a flow cell for multiplexed sequencing.