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Sample GSM1827869 Query DataSets for GSM1827869
Status Public on Jul 17, 2016
Title Pitx2-null_Input
Sample type SRA
 
Source name Forelimb
Organism Mus musculus
Characteristics antibody: none, Input
Treatment protocol Biopsies from embryos were cross-linked for 20 min in freshly made fixation buffer (1% formaldehyde 100mM NaCl, 0.5mM EGTA, 50mM HEPES, pH 8.0) and quenched by the addition of glycine (125mM). Tissue was washed twice with ice cold PBS and snap-frozen in liquid nitrogen prior to sonication (14 cycles of 10 sec at 20% output with 60 sec on ice between cycles, Branson 450 sonicator) in lysis buffer (1% SDS, 10mM EDTA pH 8.0, 50mM Tris-HCl pH 8.0, protease inhibitors) to achieve an average sheared fragment size of 300bp, with the primary smear between 200–450bp. A portion of sheared chromatin from each preparation was reserved as the input control
Growth protocol Mice heterozygous for a null allele of Pitx2 were bred and females checked for the presence of a vaginal plug (E0.5). Multiple litters, each containing 6-15 embryos were isolated at E12.5, and Pitx2-null embryos were visually identified by phenotype. The forelimbs were dissected in ice cold 1x phosphate buffered saline (PBS) and pooled with at least 10 sets per 1.5mL tube; then fixed.
Extracted molecule genomic DNA
Extraction protocol Protein G magnetic beads (NEB S1430S) were prepared by washing 3x in PBS with 0.05% bovine serum albumin (BSA), bound overnight to rabbit anti-H3K4me3 (Abcam, ab1342), rabbit anti-H3K27me3 (Millipore, 07-449), or mouse anti-Pol2 (Millipore, 17-672) at 2 µg/20 µl beads, and washed 3x in PBS-BSA. Material for immunoprecipitation (IP) as diluted 1:10 in dilution buffer (1% Triton X-100, 150mM NaCl, 2mM EDTA pH 8.0, 20mM Tris-HCl pH 8.0, protease inhibitors), and 20 µl of Protein G magnetic beads were added to each ml of sheared chromatin (about 200 µg each) and allowed to rotate overnight at 4° C. The following day, samples were washed 1x in low-salt wash (1% Triton X-100, 0.1% SDS, 150mM NaCl, 2 mM EDTA pH 8.0, 10mM Tris HCl pH 8.0, protease inhibitors), 2x in high-salt wash (1% Triton X-100, 0.1% SDS, 500mM NaCl, 2mM EDTA pH 8.0, 10mM Tris HCl pH 8.0, protease inhibitors), 2x in lithium wash (100 mM Tris HCl pH 9.0, 500mM LiCl, 1% NP40, 1% deoxycholic acid), 3x in TE (10mM Tris-HCl pH 8.0, 1mM EDTA), and eluted in 100 µl of elution buffer containing RNaseA and Proteinase K (1% SDS, 100 mM NaHCO3, 10 mg/ml RNaseA, 5 M NaCl, 0.2 mg/ml Proteinase K). Elutants and respective input controls were de-crosslinked overnight, at 65° C, purified on Qiagen Miniprep columns and eluted in ddH2O. One set from each condition was reserved for qPCR validation and the remaining ChIP DNA were speed-vac dried. DNA was resuspended in 15 µl of ddH2O, quantified with the Qubit fluorometer (Invitrogen) or the NanoDrop spectrophotometer (Thermo Scientific), until at least 10 ng were collected for each sample. 
Single-end libraries were generated using the Illumina TruSeq ChIP seq library prep kit using 18 cycles for PCR sample enrichment, with the only protocol modification being size selection after fragment amplification. The library size selected was 200–300 bp in length and, after gel purification, size was confirmed with the Bioanalyzer (Agilent). Libraries were then sequenced on a 51 bp single-end run on the Illumina HiSeq 2000
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 2000
 
Data processing Primary Illumina data image analysis, base calling, and filtering was preformed by the Casava pipline (version 1.8.2, Illumina)
Alignment of raw sequence data (fastq files) to mm10 reference genome using Bowtie2 version 2.2.3 with default parameters
Conversion of aligned .sam files into .bam files using Samtools version 1.0
Peak calling, normalization per ten million mapped reads, and generation of wig files using MACS2 with the following parameters: “-f BAM -B --SPMR --broad --broad-cutoff 0.1 -g 1.87e9 -q 0.01.”
Genome_build: mm10
Supplementary_files_format_and_content: mut_H3K4_peaks.bb: Pitx2-null H3K4me3 peaks
Supplementary_files_format_and_content: mut_H3K27_peaks.bb: Pitx2-null H3K27me3 peaks
Supplementary_files_format_and_content: mut_pol2_peaks.bb: Pitx2-null RNA Pol II peaks
Supplementary_files_format_and_content: mutant_H3K4.bw: Pitx2-null H3K4me3 signal track
Supplementary_files_format_and_content: mutant_H3K27.bw: Pitx2-null H3K4me3 signal track
Supplementary_files_format_and_content: mutant_pol2.bw: Pitx2-null RNA Pol II signal track
 
Submission date Jul 20, 2015
Last update date May 15, 2019
Contact name Chrissa Kioussi
E-mail(s) chrissa.kioussi@oregonstate.edu
Organization name Oregon State University
Department College of Pharmacy
Lab Chrissa Kioussi
Street address 1601 SW Jefferson Way
City Corvallis
State/province Oregon
ZIP/Postal code 97331
Country USA
 
Platform ID GPL13112
Series (1)
GSE71128 Mapping the Chromatin State Dynamics in Myoblasts
Relations
BioSample SAMN03891584
SRA SRX1113413

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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