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Sample GSM1278371

Query DataSets for GSM1278371

Status

Public on Jul 03, 2014

Title

Mock FLAG ChIP

Sample type

SRA

Source name

E11.5 limb bud, mock ChIP

Organism

Mus musculus

Characteristics

strain/background: Swiss Webster
tissue: limb bud
age: E11.5
chip antibody: FLAG-M2 monoclonal (Sigma, F3165)

Extracted molecule

genomic DNA

Extraction protocol

Chromatin precipitation protocol was previously described (Vokes et al. 20078). Briefly, embryonic limbs were dissociated in trypsin for 5' and then crosslinked in DMEM/10%FBS/1% formaldehyde at room-temperature for 30 minutes. Cells were lysed in lysis buffer (140mM NaCl, 0.5% NP40, 0.25% Triton-X 100, 10% glycerol, 1mM EDTA, 50mM HEPES-KOH, pH7.5) at 4˚C with agitation for 10 minutes then washed with Buffer 2 (200mM NaCl, 1mM EDTA, 0.5mM EGTA, 10mM Tris-Cl pH8.0) for 10 minutes at room-temperature. Chromatin was fragmented by sonication (Branson digital sonifier with microtip) in Buffer 3 (1mM EDTA, 0.5mM EGTA, 10mM Tris-Cl pH8.0). Lysate was cleared by centrifugation and supplemented with NaCl, NaDeoxycholate, and TritonX 100 to the final concentrations of 100mM, 0.1%, and 1%, respectively. Magnetic beads (Dynal/Invitrogen, sheep anti-mouse IgG) were blocked with a blocking buffer (5mg/ml BSA in PBS) then incubated with an antibody in the blocking buffer overnight. Unbound antibody was washed away by extensive wash with the blocking buffer. Beads/antibody complex was incubated with the lysate overnight at 4°C. Beads were washed 5 times with RIPA buffer (1% NP40, 0.7% NaDeoxycholate, 0.5M LiCl, 1mM EDTA, 50mM HEPES-KOH, pH7.5). DNA was eluted with elution buffer (1% SDS, 1mM EDTA, 50mM Tris-Cl, pH8.0) at 65˚C for 15 min, and the eluate was incubated further overnight at 65˚C to reverse crosslinking. DNA was purified using PCR purification kit (Qiagen). All buffers except RIPA buffer was supplemented with protease inhibitor cocktail mix (Roche).
Libraries were prepared according to Illumina protocols.

Library strategy

ChIP-Seq

Library source

genomic

Library selection

ChIP

Instrument model

Illumina HiSeq 2000

Data processing

Base pair calling was performed using Illumina pipeline RTA Version # 1.13.48 against a phix control-generated matrix.
50 bp reads were mapped using bwa v0.6.1-r104.
Reads mapping to Ptch1 (chr13:63,112,841-64,166,828) were filtered using samtools.
Normalized wig file was generated using QuEST.
Genome_build: mm9
Supplementary_files_format_and_content: Processed data is wig file corresponding to region: chr13:63,112,841-64,166,828.

Submission date

Dec 03, 2013

Last update date

May 15, 2019

Contact name

Kevin Peterson

E-mail(s)

kevin.peterson@med.usc.edu

Phone

323-442-8077

Organization name

Eli and Edythe Broad-CIRM Center for Regenerative Medicine, University of Southern California Keck School of Medicine