Print and check the boxes as you work through the protocol.
Processing of the input sample is described at the bottom.
- Grow 2x 250 ml of yeast cells to exponential mid-log phase (OD600 1⁄4 0.6–1.0).
- Treat cells with 7 ml 37% formaldehyde for 15 min (1% f.c.), with occasional swirling every 5 min. This allows cross-linking of protein–DNA complexes.
- wash cells 3x with 50 ml of cold sterile Milli-Q (Millipore, Billerica, MA) water. Spin down cells at 2000 rpm for 2 min and discard supernatant. Resuspend the cells in 1 ml water in a 2 ml screw-cap tubes. Spin down cells at top speed for 3 min, remove the supernatant and put on ice. Add 1 ml zirconium beads. One can continue forward to cell lysis or freeze cells at -70 C for long-term storage.
- Resuspend cells in 0.4 ml lysis/IP buffer (50 mM Hepes/KOH [pH 7.5], 140 mM NaCl, 1 mM EDTA, 1% Triton X-100, and 0.1% sodium deoxycholate) with 1 mM PMSF (Fluka, Buchs, Switzerland) and protease inhibitors (one tablet of Roche Complete protease inhibitor cocktail/50 ml lysis/IP buffer) and lyse them with zirconium beads using a FastPrep Machine (MP Biomedical, Irvine, CA; five times 60 s at a speed of 6.0 m/s)
- Recover lysates in a 5-ml snap-cap tube from one 2-ml screw-cap tube by centrifugation at 1500 rpm for 3 min, add 0.5 ml of lysis/IP buffer to the microfuge tube and centrifuge again. Make up to 1mL before sonication
- Sonicate cell lysates on the Covaris E210 instrument for 15 minutes (settings: 20% / 8 intensity/ 200 cycles per burst for 15 minutes; samples are sheared in one-minute cycles).
- The sonicated lysates should be clarified twice, first by a first centrifugation in a Sorvall centrifuge for 5 min at 3000 rpm and then by a second centrifugation in Eppendorf microfuge at 14,000 rpm for 10 min.
- Save 20 µl of clarified, sonicated lysate prior to immunoprecipitation to generate input DNA for Illumina sequencing.
- Incubate lysate overnight with antibody, rotating in cold room.
- Add 150–300 µl of prewashed beads to each sample. Immunoprecipitate for 4-6 h on a rocker in the cold room.
- After incubation, wash the immunoprecipitated samples with the appropriate buffers.
Wash the immunoprecipitated samples with 1 ml of appropriate buffer for 5–10 min on a rocker in the cold room.
Wash twice with lysis/IP buffer,
once with IP/500 mM NaCl buffer (18 ml 5 M NaCl added to 232 ml of lysis/IP buffer),
twice with IP wash buffer (10 mM Tris–HCl, 0.25 M LiCl, 0.5% NP-40, 0.5% sodium deoxycholate, and 1 mM EDTA)
once with 1 TE (50 mM Tris–HCl, 10 mM EDTA, pH 8.0). Following the last wash in TE, keep some buffer to transfer beads to a 1.5-ml tube.
- Elute the immunoprecipitate from the beads by adding 100 µl 1X TE/1% SDS and incubating for 15 min at 65 C. After 10 min, mix samples briefly. Isolate the beads on magnet and transfer eluate to a new PCR tube. Add 150 µl of 1X TE/0.67% SDS to the beads and incubate for 10 min at 65 C. Separate the beads and pool with previous eluate. Reverse protein–DNA cross-links by incubating at 65 C overnight or for 6–8 h.
- Treat samples with proteinase K to remove all proteins from samples. Dilute 20 mg/ml proteinase K (Ambion, Austin, TX) 50-fold in 1X TE. Add 250 µl of diluted proteinase K solution per sample. Incubate at 50 C for 2–4 h.
- Precipitate DNA with ethanol. Add 3 µl of 20 mg/ml glycogen, 2–5 µl of pellet paint (Novagen, San Diego, CA), 4 µl of 5 M LiCl, and 1 ml of 100% ethanol. Mix thoroughly and incubate at 20 C overnight or at least several hours. Put samples 1 h at 70 C. Spin in a cold centrifuge for 20 min at top speed and remove supernatant. The DNA pellet should be slightly pink due to pellet paint. Wash with 1 ml 70% ethanol for 5 min, spin in a cold centrifuge for 10 min at top speed and remove supernatant. Air dry for 10 min. Resuspend in 100 µl 1X TE.
- Purify DNA using MinElute PCR purification kit (Qiagen, Valencia, CA). We recommend processing the ChIP DNA sample in two MinElute spin columns. Elution is done in 21 ml EB per column and the two eluates from the same sample are pooled. Samples are stored in a -20 C freezer.
Input DNA preparation
Combine 230 µl of 1X TE/1% SDS to the reserved 20 µl of clarified sonicated lysate (from step 8, ChIP protocol) in a PCR tube. Reverse cross-links overnight by incubating at 65 C. Treat samples with proteinase K as described (step 13,ChIP protocol). Extract input DNA three times with phenol:chloroform:isoamyl alcohol (25:24:1) (Fluka) followed by a single extraction with chloroform alone. In each case, keep the upper aqueous phase. Precipitate DNA with ethanol by adding 50µl of 5M LiCl and 1ml of 100% ethanol to the upper aqueous phase from the last chloroform extraction. Enhance precipitation by transferring at -20 C for 1 h. Centrifuge samples at top speed for 20 min and discard supernatant. Wash with 1 ml of 70% ethanol in the cold room for 5 min, spin down DNA at top speed for 10 min, discard ethanol and air dry for 10 min. Resuspend DNA in 100 µl of 1X TE (pH 8.0). RNase-treat the input DNA sample. Add 2 ml of 10 mg/ml DNase-free RNase A (Roche, Indianapolis, IN) and incubate for 30 min at 37 C. Purify DNA using a MinElute PCR purification column (Qiagen). Elution is done in 21 ml of EB.
Library prep, sequencing and data analysis
Refer to our other posts on the topic of ChIP-Seq