"Investigation Title" "Chromatin immunoprecipitation microarray (ChIP-chip) study using human erythroleukemia cell line K-562 and H3K4me2 (ab7766); H3K4me3 (ab8580); H3ac (06-599); H4ac (06-866): Histone H2B(ab1790) and Histone H3 (ab1791) antibodies (ENCODE)."
"Experimental Design" binding_site_identification_design
"Experimental Factor Name" STRAINORLINE IMMUNOPRECIPITATE
"Experimental Factor Type" strain_or_line immunoprecipitate
"Person Last Name" Vetrie
"Person First Name" David
"Person Mid Initial"
"Person Email" scd@sanger.ac.uk
"Person Address" "Wellcome Trust Genome Campus;Hinxton; Cambridge; CB10 1SA; UK"
"Person Affiliation" "The Wellcome Trust Sanger Institute"
"Person Roles" investigator;submitter
"Quality Control Types" biological_replicate
"Public Release Date" "2006-09-01"
Comment[ArrayExpressSubmissionDate] "2006-08-18"
"Publication Author List" "Christoph M. Koch; Robert M. Andrews; Paul Flicek; Shane C. Dillon; Ula Kara�z; Gayle K. Clelland; Sarah Wilcox; David M. Beare; Joanna C. Fowler; Phillippe Couttet; Keith D. James; Gregory C. Lefebvre; Alexander W. Bruce; Oliver M. Dovey; Peter D. Ellis; Pawandeep Dhami; Cordelia F. Langford; Zhiping Weng; Ewan Birney; Nigel P. Carter; David Vetrie; Ian Dunham"
"Publication Title" "The Landscape of Activating Histone Modifications across 1% of the Human Genome."
"Experiment Description" "ENCODE ChIP-chip study using human myelogenous leukemia cell line K-562 and anti histone H3K4me2 (Abcam; ab7766); H3K4me3 (Abcam; ab8580); H3ac (Upstate; 06-599); H4ac (Upstate; 06-866); Histone H2B (Abcam: ab1790) and Histone H3 (Abcam: ab1791) antibodies. Each antibody experiment was conducted in three biological replicates, with two technical replicates performed for each biological replicate"
"Protocol Name" GROW_K-562 CROSSLINK_K-562_1 CROSSLINK_K-562_2 CHROMATIN_IMMUNOPRECIPITATION HYBRIDISATION_TECAN SCANARRAYEXPRESS-2 TRANSFORMATION
"Protocol Type" grow specified_biomaterial_action specified_biomaterial_action nucleic_acid_extraction hybridization image_acquisition bioassay_data_transformation
"Protocol Description" "Cells were grown in DMEM supplemented with 10% FBS and 1% Penicillin/Streptomycin to a total number of ~ 3x10^8 cells. Dilute cell density to 3 x10^5/ml every two to three days. Split and feed 16 hours before cross-linking the cells." "Day 1 Cross linking cells and chromatin extraction/preparation
1. Grow or harvest 1x10^8 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. Remove supernatant 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 4ﾰC.
Add leupeptin, PMSF and 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 4ﾰC 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 0.1M HCl for one minute, then 0.1% 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 (Sanyo/MES Soniprep sonicator)
Amplitude(microns) 14
No. of bursts: 8
Length of burst: 30 sec
Sample cooled for 1min on ice between bursts
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 4ﾰC to pellet debris
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 4ﾰC. Use the supernatant (chromatin) to set up chromatin immunoprecipitation conditions. Supernatant can be frozen at this stage. " "Same as K-562_CHROMATIN PREPARATION_1, except cells were crosslinked with 1.35 ml formaldehyde(final concentration =1%) and 3.425 ml of glycine added to quench crosslinking. " "Immunoprecipitation Day One
Control conditions
Normal rabbit IgG: 675 ul chromatin + 675 ul 1:4 NLB:IPDB buffer + 10 ul Rabbit IgG (Upstate)
Input 270 ul of chromatin store at -20ﾰC until step 5 next day.
Immunoprecipitation conditions
Histone H3 tri methyl K4 (Abcam, ab8895): 675 ul chromatin + 675 ul 1:4 NLB:IPDB buffer + 10 ug Histone H3 trimethyl K4 antibody
Histone H3 di methyl K4 (Abcam, ab7766): 675 ul chromatin + 675 ul 1:4 NLB:IPDB buffer + 2.5 ug Histone H3 dimethyl K4 antibody
Histone H3 acetyl K9/14 (Upstate, 06-599): 675 ul chromatin + 675 ul 1:4 NLB:IPDB buffer + 10 ug Histone H3 acetyl K9/K14 antibody
Histone H4 acetyl K5/8/12/16 (Upstate, 06-866): 675 ul chromatin + 675 ul 1:4 NLB:IPDB buffer + 10 ug Histone H4 acetyl K5/8/12/16 antibody
Histone H2B (Abcam, ab1790): 675 ul chromatin + 675 ul 1:4 NLB:IPDB buffer + 10 ug Histone H2B antibody
Histone H3 (Abcam, ab1791): 675 ul chromatin + 675 ul 1:4 NLB:IPDB buffer + 10 ug Histone H3 antibody
The final concentration of the anti-serum in the immunoprecipitation conditions is usually between 0.8 to 1.6%. Incubate overnight at 4ﾰC 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 4ﾰC to remove any precipitated material. 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 4ﾰC with rotation.
2. Centrifuge the protein G-agarose beads at 13000 rpm for 20 seconds at 4ﾰC. Leave the tube for 1 min to allow the protein-G agarose pellet to settle. 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 7500 rpm (Sanyo centrifuge) for 2 minutes at 4ﾰC, leave the tubes for one minute after spinning.
3. Wash the pellet similarly, once with 750 ul 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 0.2 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 65ﾰC for 6 hours
6. Add 9 ul of proteinase K (10 mg/ml stock/ Invitrogen) and incubate overnight at 45ﾰC.
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 -70ﾰC for 30 mins.
3. Centrifuge at 13200 rpm for 20 minutes at 4ﾰC. 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. Store samples at -20ﾰC.
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 " "Tecan Encode array hybridisation (gridsize: 2x6 cm)
Equipment and reagents
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), (100 ug/ul, dissolved in water)
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 3 ul
100% EtOH (cold) 1000 ul
Mix the tube gently, cover in a rack with tin foil and precipitate the DNA for 60 min at -70ﾰC.
Spin the precipitated DNA at 14000rpm at room temperature, wash the pellet with 80% ethanol and airdry the pellet.
Dissolve the pellet with 80ul hybridisation buffer per tube. This is best done by adding the hybridisation buffer and leaving the tube for 2-3 minutes in a 70ﾰC 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 100ﾰC. Mix the sample again after 5 minutes.
b. Pulse spin the tube.
c. Place the tube into a 37ﾰC heat block and incubate for 60 minutes (keep dark).
Hybridisation
Apply 140 ul of the combined tubes 1 onto the slide in an automatic hybridisation machine . Make sure that you pipette the solution absolutely bubble-free in the hybchamber. Microarrays were hybridized on an automatic hybridisation station (HS4800, Tecan) for 45h at 37ﾰC with medium agitation, washed 10 times for 1 minute with PBS 0.05% Tween20 (BDH) at 37ﾰC, 5 times for 1 minute with 0.1x SSC at 52ﾰC, 10x 1 minutes with PBS 0.05% Tween20 at 23ﾰC, followed by a final wash with HPLC-grade water (BDH) at 23ﾰC and drying under nitrogen flow for 4 minutes." "Microarrays were scanned using a ScanArray 4000 confocal laser-based scanner (Perkin Elmer). Mean spot intensities from images were quantified using ScanArray Express (Perkin Elmer) with background subtraction. Spots affected by dust were manually flagged as ﾓnot foundﾔ and subsequently excluded from the analysis. Wiggle tracks were generated combining data of all replicates to the median using an R script (see also ftp://ftp.sanger.ac.uk/pub/encode/). Tracks can be visualised under http://www.genome.ucsc.edu/ENCODE/encode.hg17.html activating the Sanger ChIP tracks in the ENCODE Chromatin Immunoprecipitation section." "The Data of all replicates of a ChIP experiment were combined to the median using an R script (see also ftp://ftp.sanger.ac.uk/pub/encode/). It takes the ""Ch2 N Ratio of Means"" values over all technical replicates, where ""Ch2 N Ratio of Means"" equals the ratio of the enriched and non-enriched fractions (both background corrected and global normalised) and combines the values to the median. Tracks of this median data can be visualised using the UCSC genome browser under http://www.genome.ucsc.edu/ENCODE/encode.hg17.html activating the Sanger ChIP tracks in the ENCODE Chromatin Immunoprecipitation section."
"Protocol Parameters"
"Protocol Software"
"SDRF File" E-TABM-140_sdrf.txt
"Term Source Name" ArrayExpress
"Term Source File" http://www.ebi.ac.uk/arrayexpress/