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Sample GSM2242113 Query DataSets for GSM2242113
Status Public on Oct 11, 2016
Title HVnSp_H3K27me3_ChIP_Rep1
Sample type SRA
 
Source name ES cells. HV-S+ FACs sorted population
Organism Mus musculus
Characteristics cell type: mESC. HhexVenus Reporter line derived from E14Tg2a cells.
purification antibody: anti-H3K27me3 (07-449; Millipore)
Treatment protocol For cell surface marker analysis by flow cytometry, 60-90% confluent ESC cultures were trypsinized and subsequently inactivated using nine volumes of FC buffer (10% FCS in PBS). After obtaining a single cell suspension, cells were centrifuged and resuspended in Ssea1 Mouse anti-Mouse IgM-kappa primary antibody (clone MC480; Developmental Studies Hybridoma Bank; 1:1500 in FC buffer) and incubated on ice for 15 mins. After two washes in ice cold FC buffer (10% FCS in PBS), cells were incubated in alexa647-conjugated Goat anti-Mouse secondary antibody (Molecular Probes A21238; 1:1000 in FC buffer) on ice for 15 mins, washed twice in FC buffer, and resuspended in 1 μg/ml DAPI in FC buffer to label dead cells. E14 ESCs labeled with secondary antibody and DAPI were used as negative controls. Forward and side scatter were used to filter out debris and cell clumps and DAPI staining was used to remove dead cells. Cells were run through a cell strainer (Corning 352235) prior to sorting on a BD FACSAriaIII. Sorted cells were collected at 4°C in 15 ml falcon tubes (Corning 352054) containing FC buffer. Different populations used in the same experiment were collected simultaneously and when multiple tubes of sorted cells were required, full tubes were stored on ice and pooled at the end of the sorting session. Sorted cells were checked for sufficient purity (>80% of cells detected as being in the original sorting gate) by running ~1000 cells through the sorter again.
Growth protocol HV5.1 ES cells (Derived from E14 Tg2a ESCs - 129/Ola background - Canham et al., 2010) were cultured on 0.1% gelatin (Sigma G1890) coated Corning flasks in ESC medium (GMEM - Sigma G5154) supplemented with 10% fetal calf serum (FCS), 1x MEM non-essential amino acids (Life Technologies 11140-036), 0.1 μM 2-Mercaptoethanol (Sigma M6250), 2 mM L-Glutamine (Life Technologies 25030-024), 1 mM Sodium Pyruvate (Life Technologies 11360-039), and 1,000 U/ml of Lif. For passaging, 60-90% confluent culture flasks were washed in PBS, incubated for 2-3 mins at 37°C in attenuated trypsin (0.025% trypsin - (Invitrogen 15090-046), 1.27 mM EDTA and 1% Chicken serum (Sigma C5405) in PBS (Sigma D8537)), and tapped to release ESCs from the flasks. Attenuated trypsin was inactivated by adding nine volumes of ESC medium and this mixture was repeatedly pipetted to obtain a single cell suspension. ESCs were centrifuged, resuspended in ESC medium and replated onto gelatin coated flasks at a ratio of between 1:2 and 1:10, or at ~5 x 10^4 cells/cm^2. All centrifugation steps with live cells were performed at 330g for 3 min at room temperature (RT).
Extracted molecule genomic DNA
Extraction protocol Approximately 0.5-2 x 10^6 FACS purified ESCs were centrifuged at 500 g for 3 min, washed twice with RT PBS and resuspended in 200 µl of culture media. Cells were fixed by the addition of an equal volume of culture media containing 2 % methanol free formaldehyde (Thermo Scientific Pierce PN28906; final concentration of 1 %) and incubated at RT for 10 min. Fixation was stopped by 5 min incubation with 125 mM glycine at room temperature. Cells were washed in PBS prior to short-term storage at -80 °C. All of the following buffers were supplemented with the following just prior to use: 1 mM DTT and 1 x Protease inhibitors (Calbiochem - 539134-1SET). Cell pellets were resuspended in lysis buffer 1 (50 mM Tris-HCl pH 8.1, 10 mM EDTA and 20 % SDS) and incubated for 10 min at 4 °C. Lysates were diluted 1:10 in ChIP dilution buffer (0.1 % Triton X-100, 2mM EDTA, 150 mM NaCl, 20 mM and Tris-HCl pH 8.1) and sonicated with a single 30 s pulse using a soniprep 150 plus (MSE; set to 3.5) followed by a further 7 pulses using a chilled Bioruptor (Diagenode; 1 min cycles of 30 sec on / 30 sec off on ‘high’ setting at 4 °C). The sonicated extract was pre-cleared by centrifugation at 16,000 g for 10 min at 4 °C. The supernatant was transferred to a fresh tube and supplemented with BSA and triton X-100 to a final concentration of 25 mg/ml and 1 % respectively. 10 % of the IP volume of the chromatin was retained as an input reference. Anti-H3K27me3 antibody (Millipore 07-449) was pre-coupled to protein A Dynabeads (Life Technologies; 10001D) at a ratio of 1 µg antibody per 5 µl of dynabeads. Antibody-bound beads were added to the fragmented chromatin at 1 µg antibody per million cell equivalents and incubated for 10 h on a rotating wheel at 4 ºC. Bead-associated immune complexes were washed once with IP buffer, three times with wash buffer 1 (150 mM NaCl, 10 mM TrisHCl pH 8, 2 mM EDTA, 1% NP40 and 1 % sodium deoxycholate) and twice in 1x TE buffer (10mM Tris pH8 and 10mM EDTA). Each wash was performed at 4ºC on a rotating wheel for 10 min. Chromatin was released from the beads by incubation with elution buffer (0.1 M NaHCO3 and 1 % SDS) for 15 min at 37 ºC followed by the addition of RNaseA and Tris pH 6.8 (final concentration of 20 mg/ml and 100 mM respectively) and incubation at 65ºC for 2 hours followed by the addition of 50 µg of proteinase K and incubation at 65ºC for a further 4-6 h to degrade proteins and reverse the cross-links. Dynabeads were removed using a magnetic rack and the chromatin purified using PCR Purification columns (Qiagen) according to manufacturer’s instructions.
Libraries were prepared as previously described (Bowman et al., 2013 - PMID: 23837789) with the following modifications. No purification was performed between the A-tailing reaction and the ligation reactions; instead the ligation was supplemented with ligation reagents (400 U of T4 DNA ligase (NEB), 1 x buffer 2 (NEB), 7.5 % PEG-6000, 1 mM ATP and 13.3 nM of annealed Illumina adaptors (AU)) and incubated at 25 °C for 90 min. Size selection purifications following the ligation and amplification PCR steps were performed with 1x and 0.8 x reaction volumes of Agencourt AMPure XP beads (Beckman Coulter - A63880). Library amplification was performed using Illumina index primers and amplified with NEBNext High-Fidelity 2X PCR Master Mix (M0541S). 4 or 5 samples were multiplexed per single Illumina HiSeq 4000 lane. libraries were seqenced as 50bp single end reads on an Illumina HiSeq 4000.
 
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina HiSeq 4000
 
Description HVnSp_IPvIN_Rep1.bw
Data processing Illumina RTA software (v2.7.3) was used for basecalling and quality control.
Illumina BCL2 FASTQ (v2.16) was used to convert .bcl files into .fastq files and demultiplex the sequence data.
bowtie2 (version 2.2.9 for windows) was used to align the reads to the mm9 mouse genome using standard paramaters.
The obtained SAM files were processed using HOMER v4.8.3. HOMER tag directories were created using the ‘makeTagDirectory’ tool with the options -unique and -fragLength 150. The HOMER function ‘removeOutOfBoundsReads.pl’ was used to remove tags which overlapped the ends of the chromosome to prevent downstream processing problems. These tag directories were used to create bedgraphs using the ‘makeUCSCfile’ tool with the following options: -pseudo 1 -norm 1e8 -avg -log -fragLength 300 -inputFragLength 300. The –i option was used to indicate the input directory with which to compare its respective IP directory. Bedgraphs were subsequently converted into browser viewable bigwig (.bw) files using the ‘bedGraphToBigWig’ utility in UCSC hosted toolset (userApps.v333; http://hgdownload.soe.ucsc.edu/admin/exe/).
Genome_build: mm9
Supplementary_files_format_and_content: bigWig genome browser file
 
Submission date Jul 19, 2016
Last update date May 15, 2019
Contact name Rob S Illingworth
E-mail(s) robert.illingworth@ed.ac.uk
Phone 01316519640
Organization name The University of Edinburgh
Department Centre for regenerative Medicine
Lab Illingworth
Street address Centre for Regenerative Medicine
City Edinburgh
State/province Midlothian
ZIP/Postal code EH16 4UU
Country United Kingdom
 
Platform ID GPL21103
Series (2)
GSE65289 Polycomb Underlies Transcriptional Heterogeneity in Lineage Priming of Embryonic Stem Cells
GSE84576 Polycomb Underlies Transcriptional Heterogeneity in Lineage Priming of Embryonic Stem Cells [ChIP-Seq]
Relations
BioSample SAMN05416651
SRA SRX1960574

Supplementary data files not provided
SRA Run SelectorHelp
Processed data are available on Series record
Raw data are available in SRA

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