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Platform GPL5955

Query DataSets for GPL5955

Status

Public on Apr 01, 2008

Title

MaPTOL for REST

Technology type

other

Distribution

virtual

Organism

Manufacturer

Scripps Research Institute

Manufacture protocol

Chromatin was prepared from mouse E12.5 embryos, sheared by sonication, and precleared with Protein-G agarose beads according to {Weinmann, 2002 #6}. An antibody (REST-H290 Ð Santa Cruz) targeted against the N-terminal region of mouse REST was used for immunoprecipitation under standard ChIP conditions {Weinmann, 2002 #6} with the following modifications. Two rounds of immunoprecipitation were carried on successively. During the second ChIP, after washing the bead-chromatin complex, they were equilibrated in NEB restriction enzyme buffer 4 with 0.5 % NP40 to sequester SDS. 80 µl of beads-chromatin slurry were then digested for 6hrs with 80 units of NlaIII. This step facilitates cloning of the co-immunoprecipitated DNA fragment and also provides footprint information as proteins that are bound to the DNA may prevent cleavage. After digestion, the complexes were again washed, eluted, and crosslinks reversed according to standard protocols {Weinmann, 2002 #6}. The entire eluate was cloned into the tagging vector pTagBM that contains two head to head MmeI restriction sites flanking the SphI cloning site. This initial library was amplified in bacteria (ECLONI Ð Lucigen) and purified using Invitrogen MaxiPrep Filter kit. 5 µg of this library was digested by 192 U of MmeI restriction enzyme overnight. After inactivation of the enzyme, we blunted the cut plasmid by incubation with 6U of T4 DNA polymerase and 10 pmol of dNTP during 3hrs at 14¼C. We stopped the reaction by addition of 10 mM EDTA for 20 min at 75¼C. The cut library was then purified by separation on an agarose gel and gel extraction using Qiagen Gel Extraction kit. 50 ng of the library was religated by overnight incubation at 14¼C with 1 µl of Invitrogen T4 DNA ligase. The religated library was used to transform E. coli competent cells (ECLONI Ð Lucigen). After quality control for religation by PCR, the library was amplified by growth on LB agar dishes (25x25 cm) and purified using Invitrogen MaxiPrep Filter kit. 100 µg of the library was digested overnight by 500 U of BamH1 restriction enzyme which sites encompass the two Mme1 sites on the pTagBM vector. The MaPTags extracted during this process were purified on an 8% polyacrylamide-TBE gel and electro-eluted from the gel (BioRad). 10 µg of MaPTags were subjected to sequencing.

Support

other

Contributor(s)

Harismendy O, Iacovoni J, Meech R

Submission date

Oct 02, 2007

Last update date

Apr 01, 2008

Contact name

Olivier Harismendy

Organization name

The Scripps Research Institute

Department

Neurobiology

Lab

Meech

Street address

10550 North Torrey PInes Road

City

San Diego

State/province

CA

ZIP/Postal code

92037

Country

USA

Samples (1)

Series (1)

MaPTOL on MmREST

Data table header descriptions
ID
CHR	UCSC.mm8.chr
BEG	UCSC.mm8.beg
END	UCSC.mm8.end
GB_RANGE
SPOT_ID

Data table
ID	CHR	BEG	END	GB_RANGE	SPOT_ID
CATGAAGGGAACTGTGACTATGTATGTAGCATG	chr1	3063747	3063972	NC_000067.4[3063747..3063972]
CATGATGCTGATCTCGTGCAGGCTGTGCAAGCCATG	chr1	3104547	3105146	NC_000067.4[3104547..3105146]
CATGAATCCAGAGATAGCCAGCAGATCACAACATG	chr1	3111562	3111740	NC_000067.4[3111562..3111740]
CATGAGAGGGGCTGAAGGAAGGAGAGCCCACTCATG	chr1	3209660	3209810	NC_000067.4[3209660..3209810]
CATGATATGTGGTCACTTTTACCATATATAATCATG	chr1	3242262	3242418	NC_000067.4[3242262..3242418]
CATGAATCTGTCTATGTTCGATTCACAAATACATG	chr1	3362534	3362719	NC_000067.4[3362534..3362719]
CATGCTCAGTAAATGTTTGACCCTAATTAGAACATG	chr1	3395222	3395298	NC_000067.4[3395222..3395298]
CATGATCTTCAAGTGGTCAGCACATATATCACATG	chr1	3475174	3475353	NC_000067.4[3475174..3475353]
CATGCTTATAGTGCAGGTGTGTGACATATGCCCATG	chr1	3528663	3528838	NC_000067.4[3528663..3528838]
CATGAGTTTATCTCTTTCAATCTACACAAAACCATG	chr1	3655763	3655931	NC_000067.4[3655763..3655931]
CATGAAGAGCTCTGATTGAGCGCAGGTGGACCATG	chr1	3720090	3720249	NC_000067.4[3720090..3720249]
CATGATGATTCAAGTGATTTCAAAGTAGTGTGCATG	chr1	3776203	3776287	NC_000067.4[3776203..3776287]
CATGATTCGGGAAGCGCCTTTTCTCCAAGGCCATG	chr1	3826307	3826452	NC_000067.4[3826307..3826452]
CATGAAACATTAAATATACGAGAAAATGTGACATG	chr1	3832047	3832244	NC_000067.4[3832047..3832244]
CATGGAGGTAGACTTCCCCCAGAAATGCTAACATG	chr1	4008762	4008970	NC_000067.4[4008762..4008970]
CATGCCAGAGAAGTCACTAATATATGGCAGCCATG	chr1	4020044	4020148	NC_000067.4[4020044..4020148]
CATGGTGTCTTCAGAAGCGTGTGAGAAGATGACATG	chr1	4050878	4051197	NC_000067.4[4050878..4051197]
CATGGGATGGAGAGCTGCATTAAACTACTACACATG	chr1	4126241	4126329	NC_000067.4[4126241..4126329]
CATGAGCTCTGGTCTATGAGCTTTAAGGAGCATG	chr1	4129278	4129395	NC_000067.4[4129278..4129395]
CATGCTGAAGCACTGGGAGGATGGAGAGTGCCATG	chr1	4249820	4250146	NC_000067.4[4249820..4250146]

Total number of rows: 49833

Table truncated, full table size 4548 Kbytes.

Download family	Format
SOFT formatted family file(s)	SOFT
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Supplementary data files not provided

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