Aspirate old medium from approximately 10 × 10⁶ cells. Cross-link by adding formaldehyde directly to culture medium (without serum) to a final concentration of 1 % and incubate for 10 min on shaker.

Stop cross-linking reaction by adding 1 ml of 1.25 M glycine and incubate gently on shaker for 5 min at room temperature.

Aspirate medium and wash cells twice using ice-cold PBS.

Add 1.5 ml of ice-cold PBS with protease inhibitors, and scrape cells in 1.5 ml Eppendorf Tube.

Pellet cells for 5 min at 1,500 rpm at 4 °C. Remove supernatant and snap-freeze cell pellet or continue immediately with immunoprecipitation protocol (see Note 1 ).

Resuspend cell pellet (5 × 10⁶ cells) in 1 ml of 0.7 % SDS lysis buffer with protease inhibitors and incubate on ice for 15 min. Divide into two tubes for sonication (500 μl in each tube) (see Note 2 ).

Sonicate lysate to shear DNA. (Diagenode Bioruptor, 3 × 15 min, 30 s ON/30 s OFF; after each 15 min cycle, centrifuge sample for 10 min on 13,000 rpm, 4 °C) (see Note 3 ).

After last centrifugation, transfer the supernatant to a new 1.5 ml microcentrifuge tube. Discard pellet.

Take an aliquot of the chromatin for sonication check.

Add 5 volumes of ChIP dilution buffer with protease inhibitors to each sample.

Preclear chromatin by adding 40 μl Protein G Agarose and place it on a rotator, 4 °C for 1 h.

Pellet agarose by brief centrifugation (4,500 × g for 1 min, 4 °C).

Remove 1 % of the supernatant as input and save at −80 °C.

Add the immunoprecipitating antibody to supernatant fraction: 4 μg of the specific antibody to one tube and 4 μg of IgG (see Note 4 ).

Incubate overnight at 4 °C with rotation.

Add 60 μl of Protein G Agarose for 3 h at 4 °C with rotation (see Note 5 ).

Pellet Protein G Agarose by brief centrifugation (3,000–5,000 × g for 1 min) and remove the supernatant fraction.

Wash the Protein G Agarose-antibody/chromatin complex by resuspending the beads in 1 ml each of the cold buffers in the order listed below and incubating for 5 min on a rotating platform followed by brief centrifugation (3,000–5,000 × g for 1 min) and careful removal of the supernatant fraction:

Elute DNA–protein–antibody complexes from beads by adding 100 μl freshly prepared elution buffer and incubate for 15 min on a rotating platform. Spin at room temperature (RT) and transfer supernatant (SN) to new tubes. Repeat and combine elutions in a 1.5 ml tube (200 μl).

For input tubes, add elution buffer up to 200 μl.

To all tubes (immunoprecipitations (IPs) and inputs), add 8 μl 5 M NaCl and 1.5 μl RNase A (10 mg/ml) and incubate at 65 °C overnight to reverse the DNA–protein cross-links.

Add 1.5 μl Proteinase K and incubate at 45 °C for 2 h.

Purify using the QIAGEN MinElute PCR Purification Kit. Elute ChIP samples in 30 μl H₂O and input samples in 50 μl (see Note 6 ).

Proceed with qPCR to test if ChIP has worked. Measure sample concentrations on NanoDrop 3300 using PicoGreen (see Note 7 ).

Mix 10 ng ChIP DNA and H₂O to a final volume of 50 μl.

Add 10 μl resuspension buffer.

Add 40 μl End Repair Mix.

Pipette gently ten times to mix.

Incubate in thermocycler: 30 °C for 30 min.

Remove sample from thermocycler and place on bench at room temperature for 1 min.

Add 160 μl AMPure beads to sample and mix ten times (see Note 8 ).

Incubate 15 min at RT and then 15 min on magnetic stand.

Remove and discard 127.5 μl of the supernatant and repeat it one more time.

Keep sample in magnetic rack and add 200 μl of fresh 80 % ethanol.

Incubate for 30 s. Remove and discard all supernatant.

Repeat this wash one more time.

Let the beads dry for 2 min and then remove from magnetic stand.

Add 17.5 μl resuspension buffer to each sample, mix, and leave for 2 min at room temperature.

Place on magnetic stand for 5 min.

Transfer 15 μl supernatant to new tube.

Safe to stop!!!

Add 2.5 μl resuspension buffer to each sample.

Add 12.5 μl A-Tailing Mix to sample and mix ten times.

Incubate at 37 °C for 30 min, 70 °C for 5 min, and hold at 4 °C.

Proceed immediately to Ligate Adapters.

Add 2.5 μl resuspension buffer to each sample.

Add 2.5 μl Ligation Mix, 2.5 μl RNA Adapter Index.

Incubate at 30 °C for 10 min.

Add 5 μl Stop Ligation Buffer.

Add 42.5 μl of well-mixed AMPure XP beads and mix by pipetting up and down until beads are in a homogenous suspension.

Incubate at room temperature for 15 min.

Place on magnetic rack for at least 5 min.

Remove and discard 80 μl of the supernatant.

Keep sample in magnetic rack and add 200 μl of 80 % ethanol.

Incubate for 30 s. Remove and discard all supernatant.

Repeat steps 28 and 29 one more time. Let the beads dry for 2 min at RT.

Add 22.5 μl of resuspension buffer and pipette up and down until beads are in a homogenous suspension.

Safe to stop!!!

Mix:

Amplify with the following PCR protocol:

Add 50 μl AMPure XP beads and incubate at room temperature for 15 min.

Place on magnetic rack for 5 min.

Remove and discard 95 μl supernatant.

Wash two times with 200 μl 80 % EtOH as usual.

Add 22.5 μl resuspension buffer and mix.

Incubate at RT for 2 min.

Place in magnetic rack for 5 min.

Transfer 20 μl to a new tube.

Prepare 2 % gel (3 g agarose in 150 ml 1× TAE buffer).

Mix 5 μl 5× loading dye with 20 μl sample and ladder.

Load samples and ladder on gel, leaving at least one empty lane between samples.

Run gel 120 V for 1 h and 30 min.

Stain gel for 30 min with SYBR Gold (30 μl SYBR Gold in 150 ml 1× TAE buffer) (see Note 10 ).

Slice the sample at 250–400 bp (see Note 11 ).

Follow the instructions of MinElute Gel Extraction Kit. Incubate the gel slices in the QG solution at RT (see Note 12 ) vortexing every 2 min.

Elute in 22 μl QIAGEN EB buffer.

Transfer 20 μl of sample to new PCR tube.

Safe to stop!!

Mix:

Amplify with the following PCR protocol:

Add 50 μl AMPure XP beads and incubate at room temperature for 15 min.

Place on magnetic rack for 5 min.

Remove and discard 95 μl supernatant.