I recommend you to check this paper 

Chromatin Immunoprecipitation (ChIP) on Unfixed Chromatin from Cells and Tissues to Analyze Histone Modifications

Alexandre Wagschal, Katia Delaval, Maëlle Pannetier, Philippe Arnaud and Robert Feil

Thank you Adriana for your recommendation. I am reading this paper now. 

                After having the powder you can resuspend the powder in buffer and filter through 37-um nylon mesh and 15-um nylon mesh to get single cells, then go ahead with point 4. below

Protocol used in Hmga2 is required for canonical WNT signaling during lung development)  good luck with your experiment     

  I.         Cell Fixation-Lysis-Chromatin Isolation

1)    Add formaldehyde 37 % directly into the cell culture media to a final concentration of 1 % (for 10 mL medium 270 µL formaldehyde 37%). Mix quickly and leave at room temperature for 10 min.

2)    Quench formaldehyde with glycine at final concentration of 125 mM (542 µL glycine 2.5 M) and transfer the cell plates on ice for about 5 minutes.

From now on all the steps until the elution of immunoprecipitated DNA will be carried out on ice.

3)    Aspirate the media and wash the cells with ice cold PBS 3 times (around 20 mL PBS 5-10 minutes each time).

4)    Add 2 mL of L1 buffer keep 5 min on ice. Scrap the cells and collect them in a 15 mL falcon tube put on ice and vortex from time to time for 20 min.

5)    Spin at 960 rpm for 5 min. at 4 C

6)    Wash with 1 mL of L1 and transfer to 1,5 mL eppis. Vortex and leave on ice for 5 min.

7)    Spin at 960 rpm for 5 min. at 4 C

8)    Transfer the cytoplasmic fraction to a new 1.5 mL tube (optional)

9)    Resuspend the nuclei in 200 μL  L2-nuclear lysis buffer.

2      Chromatin Fragmentation

1)    Sonicate the chromatin. The cycle of sonication has to be optimized before (confluent 10-cm plate à 14 cycles, embryonic lungsà 12 cycles). High

2)    Spin the sample at max speed (14000 rpm) for 10 min. at 8°C.

3)    Transfer the supernatant into a new tube.

3      Chromatin IP

1)    Dilute the rest of the chromatin solution at least 1:10 with DB-dilution buffer 3 with protease and phosphatase inhibitors and PMSF (1X) and protein A or G agarose beads conjugated with salmon sperm DNA (cut the tip of the pipet) in a 2 mL eppi (cells from 10-cm plate à 200 µL agarose beads, embryonic lungs à 100 µL agarose beads) for preclearing for at least 1 hour at 4°C in rotation (8 rpm). 

2)    Spin at 1000 g for 3 min. at 4°C in a swing out rotor and carefully transfer the supernatant into a new tube. Avoid taking any agarose beads.

3)    Distribute into 2 mL eppis in equal amounts according to the number of antibodies to be used; remember to add one more sample for non-specific IgG as the negative control.

4)    Add up to 1.8 mL volume DB and about 2 µg of antibody.

5)    Rotate (8 rpm) O/N at 4°C.

6)    Add 30 µL agarose beads (prot. A beads are good to capture polyclonal antibodies, whereas prot. G beads are good for monoclonal antibodies).

7)    Rotate for at least 2 hours rotating (8rpm) at 4°C.

8)    Spin at 1000 g for 3 min. at 4°C in a swing out rotor and carefully transfer the supernatant into a new tube. Avoid taking any agarose beads.

9)    Wash the beads with 800 µL of Low Salt washing buffer 4 with protease and phosphatase inhibitors and PMSF (1X) for 1 minute without rotation.

10)Spin at 1000 g for 1 min. at 4°C in a swing out rotor and discard or aspirate the supernatant carefully to not to disturb the beads.

11)Repeat steps 10 and 11 with the same amount of High Salt 5 and LiCl washing buffers 6 and finally twice with TE buffer (2 minutes) 7 to remove salts. After the last washing step try to leave the less liquid possible.

4      Elution of DNA from the Beads and Decrosslinking

1)    Add 100 µL of DNA elution buffer (C1) 8 on the beads.

2)    Thaw the input and fill up to 100 µL volume with TE.

3)    Add 0,5 µL RNase A (1 mg/mL) and incubate at 37°C thermo shaker at 900 rpm for 1 hour.

4)    Add 2.5 µL Proteinase K (10 mg/mL) and incubate at 56°C thermo shaker at 900 rpm for 2 hours.

5)    Add NaCl to a final concentration of 0.2 M (from 4M NaCl 5 µL to 100 µL).

6)    Incubate at 65°C thermo shaker at 900 rpm O/N.

DNA purification can be done with clroform phenol precipitation, columns or chelex.

Columns

7) Purify the DNA with Qiaquick columns according to manufacturer’s instructions:

·      4 volumes of PB buffer (1200 µL) + 10 µL NaAc (3M pH 5.0) (pH indicator in the buffer has to be yellow; the amount of NaAc can be adjusted until the solution is yellow).

·      Introduce the solution to the columns. Use a centrifuge or vacuum pump.

·      Wash the columns with 800 µL buffer PE.

·      Centrifuge 1 min. at max speed to get rid of all the buffer PE and discard the collection tubes.

·      Place the columns into new 1.5 mL eppis and add at least 30 µL of elution buffer (Tris pH 8.0) in the center of the column.

·      Incubate 5 min. at RT and then spin at max speed.

·      Measure DNA concentration with nanodrop.

·      Dilute the eluted DNA 1:4 with MQ H2O before proceeding with qPCR and store at -20°C after use. Inputs should be diluted further for qPCR reaction (an additional 1:8 or 1:16 is suggested).

P7. Eluted DNA is then subject to QPCR

Data is presented as a percentage of input (% of Input) 

ΔCt (normalized to the input samples) value for each sample

ΔCt [normalized ChIP] = (Ct [IP] - (Ct [Input] Log2 (Input Dilution Factor). X 100

Input % = 100/2 ΔCt [normalized ChIP]

The “Input %” value represents the enrichment of certain protein on specific DNA region.

Buffers:

1)    L1 lysis buffer:  50 mM Tris.HCl pH 8.0, 2 mM EDTA pH 8.0, 0.1 % NP40,

10 % glycerol

2)    L2 nuclear resuspension buffer:  50 mM Tris.HCl pH 8.0, 5 mM EDTA pH 8.0, 1 % SDS

3)    DB-dilution buffer: 50 mM Tris.HCl pH 8.0, 5 mM EDTA pH 8.0, 0.5 % NP40, 0.2 M NaCl

4)    Low Salt washing buffer: 20 mM Tris.HCl pH 8.0, 2 mM EDTA pH 8.0,

1 % NP40, 0.1 % SDS, 0.15 M NaCl

5)    High Salt washing buffer: 20 mM Tris.HCl pH 8.0, 2 mM EDTA pH 8.0,

1 % NP40, 0.1 % SDS, 0.5 M NaCl

6)    LiCl washing buffer: 10 mM Tris.HCl pH 8.0, 1 mM EDTA pH 8.0, 1 % NP40, 1% Na-deoxycholate, 0.25 M LiCl

7)    TE buffer: 10 mM Tris.HCl pH 8.0, 1 mM EDTA pH 8.0

8)    C1 DNA elution buffer: 10 mM Tris.HCl pH 8.0, 1 mM EDTA pH 8.0,

0.1 M NaHCO3, 1 % SDS

9)    DNA elution TE buffer: 25 mM Tris.HCl, 1 mM EDTA pH 10.0

Fine. Do you have any idea for ChIP-Seq? Your experience may help me. 

this protocol can be used for sending for sequecing your DNA material after IP, you require at least 1ng of DNA, better 10 ng. 

something very important that I forgot to tell you, is that you should sonicate your chromatin a lot, until you get your fragments between 200-400 bp. This is very important. Otherwise library preparation cannot be made. 

Thanks you very much for this important poiint. I am optimizing condition for sonication from muscle tissue now. I pay special attention to the fragment size rang from 200-400bp.

Active-motif kits have standardized protocols for tissues depending on the chromatin availability. Works extremely well and also can be paired up with Illumina sequencing platforms.

To sonicate the samples, I would suggest to use Bioruptor instead of micro tip sonicators. Biorupter is easy to use, you can use high power with 30 sec ON and 30 sec OFF for 10 cycles. Collect 25 ul to check fragment size after each 10 cycles and you can go for 10,20,30,40... cycles, until you get fragment size between 200-400bp.

I've recently made few ChIP-Seq libraries so if you have any other question, let me know.

good luck..!

Thanks for your your valuable suggestions. Recently I downloaded Active-motif kits protocol. Last week I discussed with my PI about the kit and she is also happy about it. 

Recently, our department installed Bioruptor sonicator. I am goiing to use it soon. Here, I am going to use same tissue for microarray as well for ChIP-Seq, the problem is the tissue size is too small (quadriceps muscle weight is around 350 mg). So, I am set up the conditions with unwanted tissue.

Thank you very much...