DNase-chip: A High Resolution Method to Identify DNaseI Hypersensitive Sites using Tiled Microarrays
Related Journal & Article Information
Journal: Nature Methods
Article Title: DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays
Introduction
Mapping DNaseI hypersensitive (HS) sites is an accurate method of identifying the location of gene regulatory elements, including promoters, enhancers, silencers, and locus control regions. While Southern blots have been the traditional method of identifying DNaseI HS sites, the conventional manual method is not readily scalable to studying large chromosomal regions, much less the entire genome. Here we describe DNase-chip, an approach that can rapidly identify DNaseI HS sites for any region of interest, or potentially for the entire genome, by using tiled microarrays. We have used DNase-chip to identify DNaseI HS sites accurately from a representative 1% of the human genome in both primary and immortalized cell types. We find that although most HS sites are present in both cell types studied, some are cell type specific. This method can be applied globally or in a targeted fashion to any tissue from any species with a sequenced genome.
Materials
Reagents
Dynal Streptavidin beads (Dynal M-280)
Dynal magnet (MPC-S cat# 120.20)
Equipment
Procedure
Blunt end DNased DNA in-gel
(For low melt gel preparation of DNase treated DNA, see Crawford, et al., Genome Research 2006)
1) Wash extensively in T4 DNA Polymerase Buffer (+DTT) , 3 × 50mls for 1 hour each (to get rid of EDTA in plug)
2) Remove all liquid from 50 ml conical tube and add Polymerase mix: 80µl DNA plug (low melt gel); 12µl 10x Polymerase Buffer (NEB#2); 5µl dNTPs (10uM; NEB Cat # M0203L); 6µl T4 DNA Polymerase ; 99.2µl H20; 2µl BSA (100x)
3) Incubate at room temp for 3-4 hours (gently mix every hour or so)
4) Add plug to 500 μl TE
5) Heat to 65 degrees for 10 minutes (flick every couple of minutes to dissolve agarose)
Phenol: Phenol/Chloroform: Chloroform extract (using wide-bore tips)
6) Add 1/10 volume 3M NaOAc, 2 volumes ETOH, and 1µl glycogen (Roche Cat# 901393)
7) Place in freezer for at least 30 minutes
8) Spin 15 minutes at 4 deg and wash 1x with 70% ETOH
9) Remove, quick spin, and remove remainder of liquid
10) Air dry for 5 minutes (NO LONGER!)
11) Resuspend in 40 μl TE
Ligation of biotinylated linkers
12) Incubate 2µg DNA, 10µl 5x Ligase Buffer (use wide bored tips; Invitrogen cat # 46300-018), 6.7µl Annealed linkers (102B/103A; THIS IS OLIGO SET A... THAW LINKERS ON ICE), 0.5µl T4 ligase (NED Cat # M0202L), H2O to 50µl at 16oC overnight.
13) Shear DNA by adding ligation to 1.5 mls TE (in 15 ml conical), putting on ice and sonicating on setting 3 for 25 second pulses (sonicate while tube is in ice bath)
14) Pulse each sample 8 times (back on ice after each sonication). The tip should be almost at the bottom of tube, with no movement. Cool the sonicator tip after each round with an ice bath
Bind to Streptavidin beads
15) Use 100 ul of beads per reaction
16) FIRST Wash beads 3× 1ml in binding/wash buffer (TE plus 1M NaCl)
17) Add 300 μl 5M NaCl to sonicated DNA (final concentration of 1M NaCl)
18) Add 100 μl of beads to sonicated DNA
19) Rock for 15 minutes at room temp
20) Use magnet to capture biotinylated ends
21) Wash 3 × 1ml in binding/wash buffer (TE plus 1M NaCl)
22) Resuspend beads in blunt ending mix (see next section)
Blunt end sheared ends
23) Incubate 97.3μl H2O, 11μl T4 Buffer (NEB buffer 2), 1 μl 10mM dNTPs (Roche # 11 814 362 001), 0.2μl T4 DNA Polymerase (NEB Cat # M0203L) and 0.5μl 100x BSA for 1 hour at 16 degrees (resuspend beads once during incubation)
24) Wash beads 3 × 1ml in binding/wash buffer (TE plus 1M NaCl)
25) Resuspend beads in 50 μl ligation mix
Ligation of non-biotinylated linkers to beads
26) Mix 32.8μl H20, 10 μl 5x Ligase Buffer (use wide bore tips; Invitrogen cat # 46300-018), 6.7μl Annealed linkers (102C/103B; THIS IS OLIGO SET C...THAW LINKERS ON ICE) and 0.5μl T4 Ligase (NEB Cat # M0202L).
27) Add mix directly to beads and resuspend
28) Incubate at 16 degrees O/N (resuspend beads once during incubation)
Clean Streptavidin beads
29) Wash 3 × 1ml in binding/wash buffer (TE plus 1M NaCl)
30) Resuspend pellet in 50 μl TE pH 8.0
LM-PCR Reaction
31) Mix 32.5μl H20, 4 μl 10x ThermoPol Buffer, 1.25μl dNTPs (10uM; Roche # 11 814 362 001), 1.25μl oligo OJW102 (40μM, 102C) and 1μl beads.
32) Place in a PCR machine and heat up the PCR machine to 50 deg, put on hold and add the following: 0.5 μl Taq DNA Polymerase 0.5 (Invitrogen cat# 18038-042), 8.5μl H20, and 1μl ThermoPol Buffer 1 (NEB Cat #B90045)
33) Cycle using the following conditions:
55 × 4 min (add Taq at this stage - hot start)
72 × 3 min
95 × 2 min
25 X (95 × 30 sec, 60 × 30 sec, 72 × 1 min)
72 × 5 min
4 x forever
Clean up of LM-PCR product
34) Add 400μl TE to pcr product
35) Do a phenol/chloroform extraction using phase locks 2.0 ml
(Brinkmann Instruments. Description: Eppendorf phase lock 2ml Cat # 62111-400)
36) ETOH ppt with 1μl glycogen (Roche Cat# 901393)
37) Resuspend pellet in 25 μl TE
38) Clean up on ArrayIT columns microarray probe purification kit (Cat #FPP $70 for 50 columns)
39) Elute with 50 μl water
Quantitate, label, and hybridize to arrays
Use the following Oligos:
1) oJW102A: 5’ biotin TEG- GCG GTG ACC CGG GAG ATC TGA ATT C –3’
2) oJW102B: 5’ /5Bio/ GCG GTG ACC CGG GAG ATC TGA ATT C –3’
3) oJW102C: 5’ GCG GTG ACC CGG GAG ATC TGA ATT C –3’
4) oJW103A: 5’ /5Phos/GAA TTC AGA TC/3AmM/ -3’
5) oJW103B: 5’ GAA TTC AGA TC -3’
Oligo SET A: 102B/103A
Oligo SET C: 102C/103B
40) Anneal primers by mixing 50 μl Tris-HCL 1M, pH 7.6; 375 μl of 40 μM oJW102; 375 μl of 40 μM oJW103 and 200 μl H2O and putting the mix in boiling water, shutting off heat, and letting oligos sit in water until room temp is reached.
41) Put at 4 degrees O/N. Aliquot and store at -20. To prevent primers from becoming un-annealed, always thaw annealed primers on ice.
Troubleshooting
Critical Steps
Anticipated Results
References
Acknowledgements
Keywords
DNaseI, Hypersensitive, Tiled Microarrays, Chromatin accessibility, DNase hypersensitive site, Nucleosome, DNase-chip