16 protocols
AccessionNameType
P-MEXP-7423
labeling
Equipment and reagents
* BioPrime Labelling Kit (Invitrogen 18094-011)
* 10X dNTP mix (1 mM dCTP, 2 mM dATP, 2 mM dGTP, 2 mM dTTP in TE buffer)
* 1mM Cy3-dCTP (Amersham Biosciences, PA55321)
* Micro-spin G50 columns (Pharmacia Amersham, 275330-01)

Method
For one Encode array two labelling reactions of Input DNA and two labelling reactions of ChIP DNA were necessary.

1. Random label 20 ul of ChIP DNA (50 ul Stock) in a final reaction volume of 150 ul by mixing the DNA with 60 ul 2.5X Random Primers Solution and make up to 130.5 ul with water.
2. Denature DNA in a heat block for 10 min at 100 C, and immediately cool on ice.
3. The following reagents are added on ice:
15 ul 10X dNTP mix
1.5 ul Cy3 labelled dCTP (1 mM)
3 ul Klenow Fragment.
Mix gently but thoroughly.
4. Incubate the reaction at 37 C overnight and stop it by adding 15 ul of stop buffer.

Removal of Labelled Nucleotides from DNA Labelling Reactions
(The recommended volume for application to a single column is 50 ul, you therefore have to use 3 columns per 150 ul labelling reaction)

Column Preparation
1. Resuspend the resin in the columns by gentle vortexing.
2. Loosen the cap one-fourth turn and snap off the bottom closure.
3. Place the columns in a 1.5 ml screw-cap microcentrifuge tube for support. Alternatively, cut the cap from a flip-top tube and use this tube for support.
4. Pre-spin the columns for 1 minute at 735 x g (4000 rpm for a MSE Micro Centaur microcentrifuge). Start the timer and microcentrifuge simultaneously. DO NOT pulse as this will override the variable speed setting. Use columns immediately after preparation to avoid the resin drying out.

Sample Application
1. Place the columns in a new 1.5 ml tube and slowly apply 50 ul to the centre of the angled surface of the compacted resin bed of each of the columns, being careful not to disturb the resin bed. Careful application of the sample to the centre of the bed is essential for good separation. Do not allow any of the samples to flow around the sides of the bed.
2. Spin the columns for 2 minutes at 735 x g (4000 rpm for a MSE Micro Centaur microcentrifuge). The purified samples are collected in the bottom of the support tube.
3. Discard the columns and retain the flow-through samples.
4. Combine the three samples and run 5 ul on a 1% agarose gel
P-MEXP-7422
labeling
Equipment and reagents
* BioPrime Labelling Kit (Invitrogen 18094-011)
* 10X dNTP mix (1 mM dCTP, 2 mM dATP, 2 mM dGTP, 2 mM dTTP in TE buffer)
* 1mM Cy5-dCTP (Amersham Biosciences, PA55321)
* Micro-spin G50 columns (Pharmacia Amersham, 275330-01)

Method
For one Encode array two labelling reactions of Input DNA and two labelling reactions of ChIP DNA were necessary.

1. Random label 0.45 ng Input DNA in a final reaction volume of 150 ul by mixing the DNA with 60 ul 2.5X Random Primers Solution and make up to 130.5 ul with water.
2. Denature DNA in a heat block for 10 min at 100C, and immediately cool on ice.
3. The following reagents are added on ice:
15 ul 10X dNTP mix
1.5 ul Cy5 labelled dCTP (1 mM)
3 ul Klenow Fragment.
Mix gently but thoroughly.
4. Incubate the reaction at 37 C overnight and stop it by adding 15 ul of stop buffer.

Removal of Labelled Nucleotides from DNA Labelling Reactions
(The recommended volume for application to a single column is 50 ul, you therefore have to use 3 columns per 150 ul labelling reaction)

Column Preparation
1. Resuspend the resin in the columns by gentle vortexing.
2. Loosen the cap one-fourth turn and snap off the bottom closure.
3. Place the columns in a 1.5 ml screw-cap microcentrifuge tube for support. Alternatively, cut the cap from a flip-top tube and use this tube for support.
4. Pre-spin the columns for 1 minute at 735 x g (4000 rpm for a MSE Micro Centaur microcentrifuge). Start the timer and microcentrifuge simultaneously. DO NOT pulse as this will override the variable speed setting. Use columns immediately after preparation to avoid the resin drying out.

Sample Application
1. Place the columns in a new 1.5 ml tube and slowly apply 50 ul to the centre of the angled surface of the compacted resin bed of each of the columns, being careful not to disturb the resin bed. Careful application of the sample to the centre of the bed is essential for good separation. Do not allow any of the samples to flow around the sides of the bed.
2. Spin the columns for 2 minutes at 735 x g (4000 rpm for a MSE Micro Centaur microcentrifuge). The purified samples are collected in the bottom of the support tube.
3. Discard the columns and retain the flow-through samples.
4. Combine the three samples and run 5 ul on a 1% agarose gel
P-MEXP-7426
bioassay_data_transformation
The median of "N Ratio of Means" values over all technical replicates (raw data files) was calculated, where "N Ratio of Means" = enriched/unenriched (both background corrected) global normalised

Erroneous features were ommitted from the transformation.
P-MEXP-7398
compound
Precleared chromatin from 4.6e6 cells is reverse crosslinked, and protein digested with proteinase K. Control DNA is extracted with phenol chloroform.
P-MEXP-7424
hybridization
Tecan Encode array hybridisation (gridsize: 2x6 cm)


Equipment and reagents

* Pre-/Hybridisation buffer: 50 % formamide
5 % dextran sulphate
0.1 % Tween 20
2x SSC
10 mM Tris pH 7.4
* 3 M NaAc pH 5.2
* human Cot1 DNA (Invitrogen, order. No. 15279011, or Roche, order. No. 1581074, test the quality prior to use)
* Herring sperm DNA (Sigma, order. No. D7290)
* 100 % Ethanol, 80 % Ethanol
* Yeast tRNA (Invitrogen, order. No. 15401-029), (10 ug/ul, dissolved in H2O)


Method

Precipitation
2 labelling reactions are needed for the ENCODE array, so make up Tube 1 twice.

* Tube 1: Cy3 labelled DNA 180 ul
Cy5 labelled DNA 180 ul
human Cot1 DNA 135 ul
3M NaAc pH 5.2 55 ul
yeast tRNA 6 ul
100% EtOH (cold) 1000 ul

* Tube 2: 10 mg/ml Herring sperm DNA 80 ul
(put into a 70 C heatblock for 5 minutes before use)
human Cot1 DNA 135 ul
3M NaAc pH 5.2 23 ?l
100 % EtOH (cold) 400 ul

Mix the tubes gently, cover the rack with tin foil and precipitate at -20C overnight or for 30 min at -70C.





Pre-hybridisation
1. Pre-heat hybridisation buffer in a 70C heatblock
2. Spin the precipitated DNAs for 15 minutes at 14000 rpm at 4 C (max. speed in Eppendorf centrifuge).
3. Remove supernatant, add 500 ul 80% EtOH, and re-spin at 14000 rpm at 4C (max. speed in Eppendorf centrifuge) for 5 minutes.
4. Remove supernatant, re-spin at 14000 rpm (max. speed in Eppendorf centrifuge) for 1 minute, take supernatant off with a small tip. Repeat until the pellet is dry.
5. Resuspend the pellets of 2 labelling reactions in

Tube 1: 160 ul Hybridisation buffer (take 50 % old Hyb buffer & 50 % new Hyb buffer)

This is best be done by adding the hybridisation buffer and leaving the tube for 2-3 minutes in a 70C heatblock before resuspending the pellet. Make sure everything is resuspended properly before you carry on with the experiment.
a. Denature the tube for 10 minutes at 100 C. Mix the sample again after 5 minutes.
b. Pulse spin the tube.
c. Place the tube into a 37 C heat block and incubate for 60 minutes (keep dark).


Tube 2: 160 ul Hybridisation buffer

This is best be done by adding the hybridisation buffer and leaving the tube for 2-3 minutes in a 70 C heatblock before resuspending the pellet. Make sure everything is resuspended properly before you carry on with the experiment.
a. Denature the tube for 10 minutes at 70 C. Mix the sample again after 5 minutes.
b. Pulse spin the tube.
c. Apply 140 ul of the denatured Herring sperm/Cot1 mix onto the slide in the Tecan. Make sure that you pipette the solution absolutely bubble-free in the hybchamber.



Pre-hybridisation

Apply 140 ul of the Tube 1 onto the slide in the Tecan. Make sure that you pipette the solution absolutely bubble-free in the hybchamber.
P-MEXP-7420
specified_biomaterial_action
Immuno-precipitation Day One

Control conditions

Normal rabbit IgG 1.35 ml chromatin + 20 ul Rabbit IgG (Upstate)
Input 270 ul of chromatin store at -20C until step 5 next day.

Immunoprecipitation conditions

Histone H3 trimethyl K4 (Abcam) 1.35 ml chromatin + 5 ug Histone H3 trimethyl K4 antibody

The final concentration of the anti-serum in the immunoprecipitation conditions is usually between 0.8 to 1.6%. Incubate overnight at 4C with rotation.


Day two

Steps 1-4 complete on ice and do not do with input!

1. Centrifuge the samples at 13000 rpm for 5 minutes at 4C. (not always a pellet). Transfer the samples to new 1.5 ml microcentrifuge tubes and add 100ul of the homogenous protein G-agarose suspension (50 ul of the bed volume). Incubate for at least 3 hours at 4C with rotation.

2. Centrifuge the protein G-agarose beads at 13000 rpm for 20 seconds at 4C. Leave the tube for 1 min in a rack (let the protein G-agarose pellet sit down, do it after every centrifugation). Remove the supernatant and wash the pellet twice with 750 ul of IP wash buffer 1 (IPWB1) For each wash, vortex briefly and centrifuge at 4000 rpm Eppendorf 5415 centrifuge (7500 rpm Sanyo centrifuge) for 2 minutes at 4 C, leave the tubes for one minute after spinning before removing the supernatant this gives a soft pellet.

3. Wash the pellet similarly, once with 750 ul of IP wash buffer 2 (IPWB2) and twice with TE pH8.0.

Keep IPEB at room temperature (high SDS spin at RT)
4. Elute the immune complexes (DNA-protein-antibody) from the beads by adding 225ul of IP dilution buffer (IPEB). Vortex strongly and centrifuge at 4000 rpm Eppendorf 5415 centrifuge (7500 rpm Sanyo centrifuge) for two minutes. Repeat this twice and combine both elutions in the same tube.

To reverse cross-links
5. Add 0.2 ul of RNase A (10 mg/ml stock/ ICN) and 27 ul of 5M NaCl (final concentration of 0.3M) to each sample. Also add 0.1ul of RNAse A and 16.2 ul of 5M NaCl to the input sample. Incubate the samples at 65C for 6 hours

6. Add 9 ul of proteinase K (10 mg/ml stock/ Invitrogen) and incubate overnight at 45C.

Day 3 Cleaning and separating DNA and protein

Complete steps in fume hood.
1. Add 2 ul tRNA (5 mg/ml stock/ Invitrogen) immediatly before adding 500ul of phenol/chloroform (in freezer). Vortex well, centrifuge at 13200 rpm for 5 minutes at room temperature. Transfer the aqueous layer to a new 2 ml microcentrifuge tubes and repeat this step once with 500 ul of chloroform. Phenol waste is kept separate.

2. Add 5 ug of glycogen, 1 ul of tRNA (5 mg/ml stock) and 50 ul of 3M sodium acetate pH 5.2 to each sample. Vortex well and add 1.25 ml of ice cold 100 % ethanol. Precipitate at -70C for 30 mins.

3. Centrifuge at 13200 rpm for 20 minutes at 4C. Wash the pellets for 10 minutes. Resuspend the pellets in 100 ul of water for the input and 50 ul of water for the other samples.

4. Remove the supernatant and air dry the pellets for 10 minutes. Resuspend the pellets in 100 ul of water for the input and 50 ul of water for the other samples.

5. Electrophorese 5 ul of each sample on a 1% agarose 1xTBE gel to check DNA size. Quantitate DNA yield using a fluorometer. Store samples at -20C.


Buffers:

IP wash buffer 1 (IPWB1)
20 mM Tris-HCl pH 8.1
50 mM NaCl
2 mM EDTA
1% Triton X-100
0.01% SDS

IP wash buffer 2 (IPWB2)
10 mM Tris-HCl pH 8.1
250 mM LiCl (1M stock 4.239g 100 ml)
1 mM EDTA
1% NP-40
1% deoxycholic acid

IP elution buffer (IPEB)
100 mM NaHCO3
0.1% SDS

RNase A (10mg/ml), Proteinase K (10mg/ml), tRNA (5mg/ml), Glycogen
P-MEXP-7392
grow
2.5x10e6 cells growing in RPMI1640 supplemented with 15 % Fetal Calf Serum, 1% Penecillin/Streptomycin for 21 days to 2x10e8 cells in total at 37 degrees C and 5% carbondioxide.
Keep cell density at 20x10e4/ml every two to three days. Split and feed 24 hours before cross-linking the cells.
P-MEXP-7426a
 
The transformed data comprises the median of the background corrected mean intensity of the enriched fraction (sample) divided by the background corrected mean intensity of the non-enriched fraction (input) of six replicates: two technical replicates (immunoprecipitation and hybridisations) for each of the three biological replicates (independent cell cultures).
P-MEXP-8769
specified_biomaterial_action
Extraction protocol description:

11. Preclear chromatin by adding 100ul of normal rabbit IgG (Upstate) and incubate for 1 hour at 4C on a rotating wheel. Add 200 ul of the homogenous protein G-agarose suspension (100ul of the bed volume/Roche) and incubate for 3 hours (or overnight) at 4C on a rotating wheel (cold room). 12. Centrifuge the beads at 3000 rpm for two minutes at 4C. Use the supernatant (chromatin) to set up the following conditions. Supernatant can be frozen at this stage Immuno-precipitation Day One Control conditions Normal rabbit IgG 1.35 ml chromatin + 20ul Rabbit IgG (Upstate) Input 270ul of chromatin store at -20C until step 5 next day. Immunoprecipitation conditions 1.35ml chromatin + 10ug Histone H3 acetylated antibody (06-599, Upstate). The final concentration of the anti-serum in the immunoprecipitation conditions is usually between 0.8 to 1.6%. Incubate overnight at 4C with rotation. Day two Steps 1-4 complete on ice and do not do with input!

1. Centrifuge the samples at 13000 rpm for 5 minutes at 4C. (not always a pellet). Transfer the samples to new 1.5 ml microcentrifuge tubes and add 100ul of the homogenous protein G-agarose suspension (50 ul of the bed volume). Incubate for at least 3 hours at 4C with rotation.
2. Centrifuge the protein G-agarose beads at 13000 rpm for 20 seconds at 4C. Leave the tube for 1 min in a rack (let the protein G-agarose pellet sit down, do it after every centrifugation). Remove the supernatant and wash the pellet twice with 750 ul of IP wash buffer 1 (IPWB1) For each wash, vortex briefly and centrifuge at 4000 rpm Eppendorf 5415 centrifuge (7500 rpm Sanyo centrifuge) for 2 minutes at 4C, leave the tubes for one minute after spinning before removing the supernatant this gives a soft pellet.
3. Wash the pellet similarly, once with 750ul of IP wash buffer 2 (IPWB2) and twice with TE pH 8.0. Keep IPEB at room temperature (high SDS spin at RT)
4. Elute the immune complexes (DNA-protein-antibody) from the beads by adding 225ul of IP dilution buffer (IPEB). Vortex strongly and centrifuge at 4000 rpm Eppendorf 5415 centrifuge (7500 rpm Sanyo centrifuge) for two minutes. Repeat this twice and combine both elutions in the same tube. To reverse cross-links
5. Add 0.2ul of RNase A (10mg/ml stock/ ICN) and 27ul of 5M NaCl (final concentration of 0.3M) to each sample. Also add 0.1ul of RNAse A and 16.2ul of 5M NaCl to the input sample. Incubate the samples at 65C for 6 hours
6. Add 9ul of proteinase K (10mg/ml stock/ Invitrogen) and incubate overnight at 45C. Day 3 Cleaning and separating DNA and protein Complete steps in fume hood.

1. Add 2ul tRNA (5mg/ml stock/ Invitrogen) immediatly before adding 500ul of phenol/chloroform (in freezer). Vortex well, centrifuge at 13200 rpm for 5 minutes at room temperature. Transfer the aqueous layer to a new 2ml microcentrifuge tubes and repeat this step once with 500ul of chloroform. Phenol waste is kept separate.
2. Add 5 ug of glycogen, 1 ul of tRNA (5 mg/ml stock) and 50 ul of 3M sodium acetate pH 5.2 to each sample. Vortex well and add 1.25 ml of ice cold 100 % ethanol. Precipitate at -70C for 30 mins.
3. Centrifuge at 13200 rpm for 20 minutes at 4C. Wash the pellets for 10 minutes. Resuspend the pellets in 100 ul of water for the input and 50ul of water for the other samples.
4. Remove the supernatant and air dry the pellets for 10 minutes. Resuspend the pellets in 100ul of water for the input and 50ul of water for the other samples.
5. Electrophorese 5ul of each sample on a 1% agarose 1xTBE gel to check DNA size. Quantitate DNA yield using a fluorometer. Store samples at -20C.

Buffers:

IP wash buffer 1 (IPWB1)
20mM Tris-HCl pH 8.1
50mM NaCl
2mM EDTA
1% Triton X-100
0.01% SDS

IP wash buffer 2 (IPWB2)
10mM Tris-HCl pH 8.1
250mM LiCl (1M stock 4.239g 100 ml)
1 mM EDTA
1% NP-40
1% deoxycholic acid

IP elution buffer (IPEB)
100mM NaHCO3 0.1% SDS RNase A (10mg/ml),
Proteinase K (10mg/ml),
tRNA (5mg/ml),
Glycogen
P-MEXP-9223
grow
2.5x10e6 cells growing in RPMI1640 supplemented with 15 % Fetal Calf Serum, 1% Penecillin/Streptomycin for 21 days to 6-12x10e8 cells in total at 37 degrees C and 5% carbondioxide. Keep cell density at 20x10e4/ml every two to three days. Split and feed 24 hours before cross-linking the cells.
P-MEXP-9224
nucleic_acid_extraction
Day 1 Cross linking cells and chromatin extraction

1. Grow or harvest 1 x10<sup>8</sup> cells. Collect the cells by centrifuging at 1200 rpm for 5-8 minutes.
2. Resuspend the cells in pre-warmed 50 ml serum free media in a glass flask. Add 1.4 ml of formaldehyde solution in fume hood (37%; final concentration 1%) to cross link DNA-protein interactions. Incubate at room temperature with gentle agitation for 10 minutes.
3. Add 3.15 ml of 2M glycine (final concentration of 0.125M) and incubate for 5 minutes at room temperature with gentle shaking to stop the cross linking reaction.
4. Transfer the cells to a 50 ml Falcon tube on ice and centrifuge the cells at 1200 rpm for 6 minutes at 4C.
5. Discard supernatant in formaldehyde waste bottle in fume hood and resuspend the pellet with 1.5 ml ice-cold PBS and keep on ice. Centrifuge the cells at 2000 rpm for 5 minutes at 4C. Add 20ul of leupeptin and 20ul sodium butyrate to CLB immediately before use
6. Gently resuspend the cell pellet in 1.5X pellet volume (this is approximate) of cell lysis buffer (CLB). Resuspend the cells by pipetting up and down and incubate for 10 minutes on ice. Centrifuge at 2500 rpm for 5 minutes at 4C to collect nuclei. Keep NLB at room temperature
7. Remove the supernatant and resuspend the nuclei in 1.2 ml of nuclear lysis buffer (NLB) and incubate on ice for 10 minutes. Add 0.72 ml of IP dilution buffer (IPDB) and transfer to a 5 ml falcon tube.
8. Sonicate. First clean the tip with HCl for one minute, then 1% SDS then rinse with HPLC water for one minute. Keep sample on ice at all times and position the sonicator tip just below the surface and the sides of the tube not touching the sonicator. The sample should turn from cloudy to clear.

Settings (Branson 450 digital with microtip)

Time 8 min
Amplitude 16 %
Pulse on 0.5 sec
Pulse off 2.0 sec
While sonification samples were cooled in a ice water bath.

9. Using these conditions, the DNA is sheared to approximately 300-600 bp fragments. Transfer the sheared chromatin to 2ml microcentrifuge tubes and centrifuge at 13000 rpm for 5 minutes at 4 C. Remove debris and take out 10 ul aliquot for gel.
10. Transfer the supernatant to a 15 ml Falcon and add 4.1 ml of IPDB to each tube to bring the ratio of NLB:IPDB to 1:4. Supernatant can be frozen at this stage

Buffers:

cell lysis buffer (CLB)
10 mM Tris-HCl pH 8.0
10 mM NaCl 0.2% NP40
10 mM Sodium butyrate
50 ug/ml PMSF
1 ug/ml leupeptin

nuclear lysis buffer (NLB)
50 mM Tris-HCl pH 8.1
10 mM EDTA 1% SDS
10 mM Sodium butyrate
50 ug/ml PMSF
1 ug/ml leupeptin

IP dilution buffer (IPDB)
20 mM Tris-HCl pH 8.1
150 mM NaCl
2 mM EDTA
1% Triton X-100
0.01% SDS
10 mM Sodium butyrate
50 ug/ml PMSF
1 ug/ml leupeptin.
P-MEXP-8770
specified_biomaterial_action
Extraction protocol description:

11. Preclear chromatin by adding 100ul of normal rabbit IgG (Upstate) and incubate for 1 hour at 4C on a rotating wheel. Add 200 ul of the homogenous protein G-agarose suspension (100ul of the bed volume/Roche) and incubate for 3 hours (or overnight) at 4C on a rotating wheel (cold room). 12. Centrifuge the beads at 3000 rpm for two minutes at 4C. Use the supernatant (chromatin) to set up the following conditions. Supernatant can be frozen at this stage Immuno-precipitation Day One Control conditions Normal rabbit IgG 1.35 ml chromatin + 20ul Rabbit IgG (Upstate) Input 270ul of chromatin store at -20C until step 5 next day. Immunoprecipitation conditions 1.35ml chromatin + 10ug Histone H3K4me3 antibody (ab8580, Abcam). The final concentration of the anti-serum in the immunoprecipitation conditions is usually between 0.8 to 1.6%. Incubate overnight at 4C with rotation. Day two Steps 1-4 complete on ice and do not do with input!

1. Centrifuge the samples at 13000 rpm for 5 minutes at 4C. (not always a pellet). Transfer the samples to new 1.5 ml microcentrifuge tubes and add 100ul of the homogenous protein G-agarose suspension (50 ul of the bed volume). Incubate for at least 3 hours at 4C with rotation.
2. Centrifuge the protein G-agarose beads at 13000 rpm for 20 seconds at 4C. Leave the tube for 1 min in a rack (let the protein G-agarose pellet sit down, do it after every centrifugation). Remove the supernatant and wash the pellet twice with 750 ul of IP wash buffer 1 (IPWB1) For each wash, vortex briefly and centrifuge at 4000 rpm Eppendorf 5415 centrifuge (7500 rpm Sanyo centrifuge) for 2 minutes at 4C, leave the tubes for one minute after spinning before removing the supernatant this gives a soft pellet.
3. Wash the pellet similarly, once with 750ul of IP wash buffer 2 (IPWB2) and twice with TE pH 8.0. Keep IPEB at room temperature (high SDS spin at RT)
4. Elute the immune complexes (DNA-protein-antibody) from the beads by adding 225ul of IP dilution buffer (IPEB). Vortex strongly and centrifuge at 4000 rpm Eppendorf 5415 centrifuge (7500 rpm Sanyo centrifuge) for two minutes. Repeat this twice and combine both elutions in the same tube. To reverse cross-links
5. Add 0.2ul of RNase A (10mg/ml stock/ ICN) and 27ul of 5M NaCl (final concentration of 0.3M) to each sample. Also add 0.1ul of RNAse A and 16.2ul of 5M NaCl to the input sample. Incubate the samples at 65C for 6 hours
6. Add 9ul of proteinase K (10mg/ml stock/ Invitrogen) and incubate overnight at 45C. Day 3 Cleaning and separating DNA and protein Complete steps in fume hood.

1. Add 2ul tRNA (5mg/ml stock/ Invitrogen) immediatly before adding 500ul of phenol/chloroform (in freezer). Vortex well, centrifuge at 13200 rpm for 5 minutes at room temperature. Transfer the aqueous layer to a new 2ml microcentrifuge tubes and repeat this step once with 500ul of chloroform. Phenol waste is kept separate.
2. Add 5 ug of glycogen, 1 ul of tRNA (5 mg/ml stock) and 50 ul of 3M sodium acetate pH 5.2 to each sample. Vortex well and add 1.25 ml of ice cold 100 % ethanol. Precipitate at -70C for 30 mins.
3. Centrifuge at 13200 rpm for 20 minutes at 4C. Wash the pellets for 10 minutes. Resuspend the pellets in 100 ul of water for the input and 50ul of water for the other samples.
4. Remove the supernatant and air dry the pellets for 10 minutes. Resuspend the pellets in 100ul of water for the input and 50ul of water for the other samples.
5. Electrophorese 5ul of each sample on a 1% agarose 1xTBE gel to check DNA size. Quantitate DNA yield using a fluorometer. Store samples at -20C.

Buffers:

IP wash buffer 1 (IPWB1)
20mM Tris-HCl pH 8.1
50mM NaCl
2mM EDTA
1% Triton X-100
0.01% SDS

IP wash buffer 2 (IPWB2)
10mM Tris-HCl pH 8.1
250mM LiCl (1M stock 4.239g 100 ml)
1 mM EDTA
1% NP-40
1% deoxycholic acid

IP elution buffer (IPEB)
100mM NaHCO3 0.1% SDS RNase A (10mg/ml),
Proteinase K (10mg/ml),
tRNA (5mg/ml),
Glycogen
P-MEXP-8838
specified_biomaterial_action
Extraction protocol description:

11. Preclear chromatin by adding 100ul of normal rabbit IgG (Upstate) and incubate for 1 hour at 4C on a rotating wheel. Add 200 ul of the homogenous protein G-agarose suspension (100ul of the bed volume/Roche) and incubate for 3 hours (or overnight) at 4C on a rotating wheel (cold room). 12. Centrifuge the beads at 3000 rpm for two minutes at 4C. Use the supernatant (chromatin) to set up the following conditions. Supernatant can be frozen at this stage Immuno-precipitation Day One Control conditions Normal rabbit IgG 1.35 ml chromatin + 20ul Rabbit IgG (Upstate) Input 270ul of chromatin store at -20C until step 5 next day. Immunoprecipitation conditions 1.35ml chromatin + 10ug Histone H4 acetylated antibody (06-866, Upstate). The final concentration of the anti-serum in the immunoprecipitation conditions is usually between 0.8 to 1.6%. Incubate overnight at 4C with rotation. Day two Steps 1-4 complete on ice and do not do with input!

1. Centrifuge the samples at 13000 rpm for 5 minutes at 4C. (not always a pellet). Transfer the samples to new 1.5 ml microcentrifuge tubes and add 100ul of the homogenous protein G-agarose suspension (50 ul of the bed volume). Incubate for at least 3 hours at 4C with rotation.
2. Centrifuge the protein G-agarose beads at 13000 rpm for 20 seconds at 4C. Leave the tube for 1 min in a rack (let the protein G-agarose pellet sit down, do it after every centrifugation). Remove the supernatant and wash the pellet twice with 750 ul of IP wash buffer 1 (IPWB1) For each wash, vortex briefly and centrifuge at 4000 rpm Eppendorf 5415 centrifuge (7500 rpm Sanyo centrifuge) for 2 minutes at 4C, leave the tubes for one minute after spinning before removing the supernatant this gives a soft pellet.
3. Wash the pellet similarly, once with 750ul of IP wash buffer 2 (IPWB2) and twice with TE pH 8.0. Keep IPEB at room temperature (high SDS spin at RT)
4. Elute the immune complexes (DNA-protein-antibody) from the beads by adding 225ul of IP dilution buffer (IPEB). Vortex strongly and centrifuge at 4000 rpm Eppendorf 5415 centrifuge (7500 rpm Sanyo centrifuge) for two minutes. Repeat this twice and combine both elutions in the same tube. To reverse cross-links
5. Add 0.2ul of RNase A (10mg/ml stock/ ICN) and 27ul of 5M NaCl (final concentration of 0.3M) to each sample. Also add 0.1ul of RNAse A and 16.2ul of 5M NaCl to the input sample. Incubate the samples at 65C for 6 hours
6. Add 9ul of proteinase K (10mg/ml stock/ Invitrogen) and incubate overnight at 45C. Day 3 Cleaning and separating DNA and protein Complete steps in fume hood.

1. Add 2ul tRNA (5mg/ml stock/ Invitrogen) immediatly before adding 500ul of phenol/chloroform (in freezer). Vortex well, centrifuge at 13200 rpm for 5 minutes at room temperature. Transfer the aqueous layer to a new 2ml microcentrifuge tubes and repeat this step once with 500ul of chloroform. Phenol waste is kept separate.
2. Add 5 ug of glycogen, 1 ul of tRNA (5 mg/ml stock) and 50 ul of 3M sodium acetate pH 5.2 to each sample. Vortex well and add 1.25 ml of ice cold 100 % ethanol. Precipitate at -70C for 30 mins.
3. Centrifuge at 13200 rpm for 20 minutes at 4C. Wash the pellets for 10 minutes. Resuspend the pellets in 100 ul of water for the input and 50ul of water for the other samples.
4. Remove the supernatant and air dry the pellets for 10 minutes. Resuspend the pellets in 100ul of water for the input and 50ul of water for the other samples.
5. Electrophorese 5ul of each sample on a 1% agarose 1xTBE gel to check DNA size. Quantitate DNA yield using a fluorometer. Store samples at -20C.

Buffers:

IP wash buffer 1 (IPWB1)
20mM Tris-HCl pH 8.1
50mM NaCl
2mM EDTA
1% Triton X-100
0.01% SDS

IP wash buffer 2 (IPWB2)
10mM Tris-HCl pH 8.1
250mM LiCl (1M stock 4.239g 100 ml)
1 mM EDTA
1% NP-40
1% deoxycholic acid

IP elution buffer (IPEB)
100mM NaHCO3
0.1% SDS RNase A (10mg/ml),
Proteinase K (10mg/ml),
tRNA (5mg/ml),
Glycogen.
P-MEXP-8840
specified_biomaterial_action
11. Preclear chromatin by adding 100ul of normal rabbit IgG (Upstate) and incubate for 1 hour at 4C on a rotating wheel. Add 200 ul of the homogenous protein G-agarose suspension (100ul of the bed volume/Roche) and incubate for 3 hours (or overnight) at 4C on a rotating wheel (cold room). 12. Centrifuge the beads at 3000 rpm for two minutes at 4C. Use the supernatant (chromatin) to set up the following conditions. Supernatant can be frozen at this stage Immuno-precipitation Day One Control conditions Normal rabbit IgG 1.35 ml chromatin + 20ul Rabbit IgG (Upstate) Input 270ul of chromatin store at -20C until step 5 next day. Immunoprecipitation conditions 1.35ml chromatin + 10ug Histone H3 mono methyl Lysine 4 antibody (ab8895, Abcam). The final concentration of the anti-serum in the immunoprecipitation conditions is usually between 0.8 to 1.6%. Incubate overnight at 4C with rotation. Day two Steps 1-4 complete on ice and do not do with input!

1. Centrifuge the samples at 13000 rpm for 5 minutes at 4C. (not always a pellet). Transfer the samples to new 1.5 ml microcentrifuge tubes and add 100ul of the homogenous protein G-agarose suspension (50 ul of the bed volume). Incubate for at least 3 hours at 4C with rotation.
2. Centrifuge the protein G-agarose beads at 13000 rpm for 20 seconds at 4C. Leave the tube for 1 min in a rack (let the protein G-agarose pellet sit down, do it after every centrifugation). Remove the supernatant and wash the pellet twice with 750 ul of IP wash buffer 1 (IPWB1) For each wash, vortex briefly and centrifuge at 4000 rpm Eppendorf 5415 centrifuge (7500 rpm Sanyo centrifuge) for 2 minutes at 4C, leave the tubes for one minute after spinning before removing the supernatant this gives a soft pellet.
3. Wash the pellet similarly, once with 750ul of IP wash buffer 2 (IPWB2) and twice with TE pH 8.0. Keep IPEB at room temperature (high SDS spin at RT)
4. Elute the immune complexes (DNA-protein-antibody) from the beads by adding 225ul of IP dilution buffer (IPEB). Vortex strongly and centrifuge at 4000 rpm Eppendorf 5415 centrifuge (7500 rpm Sanyo centrifuge) for two minutes. Repeat this twice and combine both elutions in the same tube. To reverse cross-links
5. Add 0.2ul of RNase A (10mg/ml stock/ ICN) and 27ul of 5M NaCl (final concentration of 0.3M) to each sample. Also add 0.1ul of RNAse A and 16.2ul of 5M NaCl to the input sample. Incubate the samples at 65C for 6 hours
6. Add 9ul of proteinase K (10mg/ml stock/ Invitrogen) and incubate overnight at 45C. Day 3 Cleaning and separating DNA and protein Complete steps in fume hood.

1. Add 2ul tRNA (5mg/ml stock/ Invitrogen) immediatly before adding 500ul of phenol/chloroform (in freezer). Vortex well, centrifuge at 13200 rpm for 5 minutes at room temperature. Transfer the aqueous layer to a new 2ml microcentrifuge tubes and repeat this step once with 500ul of chloroform. Phenol waste is kept separate.
2. Add 5 ug of glycogen, 1 ul of tRNA (5 mg/ml stock) and 50 ul of 3M sodium acetate pH 5.2 to each sample. Vortex well and add 1.25 ml of ice cold 100 % ethanol. Precipitate at -70C for 30 mins.
3. Centrifuge at 13200 rpm for 20 minutes at 4C. Wash the pellets for 10 minutes. Resuspend the pellets in 100 ul of water for the input and 50ul of water for the other samples.
4. Remove the supernatant and air dry the pellets for 10 minutes. Resuspend the pellets in 100ul of water for the input and 50ul of water for the other samples.
5. Electrophorese 5ul of each sample on a 1% agarose 1xTBE gel to check DNA size. Quantitate DNA yield using a fluorometer. Store samples at -20C.

Buffers:

IP wash buffer 1 (IPWB1)
20mM Tris-HCl pH 8.1
50mM NaCl
2mM EDTA
1% Triton X-100
0.01% SDS

IP wash buffer 2 (IPWB2)
10mM Tris-HCl pH 8.1
250mM LiCl (1M stock 4.239g 100 ml)
1 mM EDTA
1% NP-40
1% deoxycholic acid

IP elution buffer (IPEB)
100mM NaHCO3
0.1% SDS RNase A (10mg/ml),
Proteinase K (10mg/ml),
tRNA (5mg/ml),
Glycogen.
P-MEXP-8845
specified_biomaterial_action
11. Preclear chromatin by adding 100ul of normal rabbit IgG (Upstate) and incubate for 1 hour at 4C on a rotating wheel. Add 200 ul of the homogenous protein G-agarose suspension (100ul of the bed volume/Roche) and incubate for 3 hours (or overnight) at 4C on a rotating wheel (cold room). 12. Centrifuge the beads at 3000 rpm for two minutes at 4C. Use the supernatant (chromatin) to set up the following conditions. Supernatant can be frozen at this stage Immuno-precipitation Day One Control conditions Normal rabbit IgG 1.35 ml chromatin + 20ul Rabbit IgG (Upstate) Input 270ul of chromatin store at -20C until step 5 next day. Immunoprecipitation conditions 1.35ml chromatin + 10ug Histone H3 dimethyl Lysine 4 antibody (ab7766, Abcam). The final concentration of the anti-serum in the immunoprecipitation conditions is usually between 0.8 to 1.6%. Incubate overnight at 4C with rotation. Day two Steps 1-4 complete on ice and do not do with input!

1. Centrifuge the samples at 13000 rpm for 5 minutes at 4C. (not always a pellet). Transfer the samples to new 1.5 ml microcentrifuge tubes and add 100ul of the homogenous protein G-agarose suspension (50 ul of the bed volume). Incubate for at least 3 hours at 4C with rotation.
2. Centrifuge the protein G-agarose beads at 13000 rpm for 20 seconds at 4C. Leave the tube for 1 min in a rack (let the protein G-agarose pellet sit down, do it after every centrifugation). Remove the supernatant and wash the pellet twice with 750 ul of IP wash buffer 1 (IPWB1) For each wash, vortex briefly and centrifuge at 4000 rpm Eppendorf 5415 centrifuge (7500 rpm Sanyo centrifuge) for 2 minutes at 4C, leave the tubes for one minute after spinning before removing the supernatant this gives a soft pellet.
3. Wash the pellet similarly, once with 750ul of IP wash buffer 2 (IPWB2) and twice with TE pH 8.0. Keep IPEB at room temperature (high SDS spin at RT)
4. Elute the immune complexes (DNA-protein-antibody) from the beads by adding 225ul of IP dilution buffer (IPEB). Vortex strongly and centrifuge at 4000 rpm Eppendorf 5415 centrifuge (7500 rpm Sanyo centrifuge) for two minutes. Repeat this twice and combine both elutions in the same tube. To reverse cross-links
5. Add 0.2ul of RNase A (10mg/ml stock/ ICN) and 27ul of 5M NaCl (final concentration of 0.3M) to each sample. Also add 0.1ul of RNAse A and 16.2ul of 5M NaCl to the input sample. Incubate the samples at 65C for 6 hours
6. Add 9ul of proteinase K (10mg/ml stock/ Invitrogen) and incubate overnight at 45C. Day 3 Cleaning and separating DNA and protein Complete steps in fume hood.

1. Add 2ul tRNA (5mg/ml stock/ Invitrogen) immediatly before adding 500ul of phenol/chloroform (in freezer). Vortex well, centrifuge at 13200 rpm for 5 minutes at room temperature. Transfer the aqueous layer to a new 2ml microcentrifuge tubes and repeat this step once with 500ul of chloroform. Phenol waste is kept separate.
2. Add 5 ug of glycogen, 1 ul of tRNA (5 mg/ml stock) and 50 ul of 3M sodium acetate pH 5.2 to each sample. Vortex well and add 1.25 ml of ice cold 100 % ethanol. Precipitate at -70C for 30 mins.
3. Centrifuge at 13200 rpm for 20 minutes at 4C. Wash the pellets for 10 minutes. Resuspend the pellets in 100 ul of water for the input and 50ul of water for the other samples.
4. Remove the supernatant and air dry the pellets for 10 minutes. Resuspend the pellets in 100ul of water for the input and 50ul of water for the other samples.
5. Electrophorese 5ul of each sample on a 1% agarose 1xTBE gel to check DNA size. Quantitate DNA yield using a fluorometer. Store samples at -20C.

Buffers:

IP wash buffer 1 (IPWB1)
20mM Tris-HCl pH 8.1
50mM NaCl
2mM EDTA
1% Triton X-100
0.01% SDS

IP wash buffer 2 (IPWB2)
10mM Tris-HCl pH 8.1
250mM LiCl (1M stock 4.239g 100 ml)
1 mM EDTA
1% NP-40
1% deoxycholic acid

IP elution buffer (IPEB)
100mM NaHCO3
0.1% SDS RNase A (10mg/ml),
Proteinase K (10mg/ml),
tRNA (5mg/ml),
Glycogen.
P-MEXP-7419
nucleic_acid_extraction
Day 1 Cross linking cells and chromatin extraction

1. Grow or harvest 1x10e8 cells. Collect the cells by centrifuging at 1200 rpm for 5-8 minutes.

2. Resuspend the cells in pre-warmed 50 ml serum free media in a glass flask. Add 0.5 ml of formaldehyde solution in fume hood (37%; final concentration 0.37%) to cross link DNA-protein interactions. Incubate at room temperature with gentle agitation for 10 minutes.

3. Add 3.15 ml of 2M glycine (final concentration of 0.125M) and incubate for 5 minutes at room temperature with gentle shaking to stop the cross linking reaction.

4. Transfer the cells to a 50 ml Falcon tube on ice and centrifuge the cells at 1200 rpm for 6 minutes at 4C.

5. Discard supernatant in formaldehyde waste bottle in fume hood and resuspend the pellet with 1.5 ml ice-cold PBS and keep on ice. Centrifuge the cells at 2000 rpm for 5 minutes at 4C.

Add 20ul of leupeptin and 20ul sodium butyrate to CLB immediately before use

6. Gently resuspend the cell pellet in 1.5X pellet volume (this is approximate) of cell lysis buffer (CLB). Resuspend the cells by pipetting up and down and incubate for 10 minutes on ice. Centrifuge at 2500 rpm for 5 minutes at 4C to collect nuclei.

Keep NLB at room temperature

7. Remove the supernatant and resuspend the nuclei in 1.2 ml of nuclear lysis buffer (NLB) and incubate on ice for 10 minutes. Add 0.72 ml of IP dilution buffer (IPDB) and transfer to a 5 ml falcon tube.

8. Sonicate. First clean the tip with HCl for one minute, then 1% SDS then rinse with HPLC water for one minute. Keep sample on ice at all times and position the sonicator tip just below the surface and the sides of the tube not touching the sonicator. The sample should turn from cloudy to clear.

Settings (Branson 450 digital with microtip)

Time 8 min
Amplitude 16 %
Pulse on 0.5 sec
Pulse off 2.0 sec
While sonification samples were cooled in a ice water bath.

9. Using these conditions, the DNA is sheared to approximately 300-600 bp fragments. Transfer the sheared chromatin to 2ml microcentrifuge tubes and centrifuge at 13000 rpm for 5 minutes at 4 C. Remove debris and take out 10 ul aliquot for gel.

10. Transfer the supernatant to a 15 ml Falcon and add 4.1 ml of IPDB to each tube to bring the ratio of NLB:IPDB to 1:4. Supernatant can be frozen at this stage

Buffers:

cell lysis buffer (CLB)
10 mM Tris-HCl pH 8.0
10 mM NaCl
0.2% NP40
10 mM Sodium butyrate
50 ug/ml PMSF
1 ug/ml leupeptin


nuclear lysis buffer (NLB)
50 mM Tris-HCl pH 8.1
10 mM EDTA
1% SDS
10 mM Sodium butyrate
50 ug/ml PMSF
1 ug/ml leupeptin

IP dilution buffer (IPDB)
20 mM Tris-HCl pH 8.1
150 mM NaCl
2 mM EDTA
1% Triton X-100
0.01% SDS
10 mM Sodium butyrate
50 ug/ml PMSF
1 ug/ml leupeptin

11. Preclear chromatin by adding 100ul of normal rabbit IgG (Upstate) and incubate for 1 hour at 4C on a rotating wheel. Add 200 ul of the homogenous protein G-agarose suspension (100ul of the bed volume/Roche) and incubate for 3 hours (or overnight) at 4C on a rotating wheel (cold room).

12. Centrifuge the beads at 3000 rpm for two minutes at 4C. Use the supernatant (chromatin) to set up the following conditions. Supernatant can be frozen at this stage.