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Sample GSM3908341 Query DataSets for GSM3908341
Status Public on Sep 11, 2019
Title TKO_Rybp
Sample type SRA
Source name Embryonic stem cell and NT2 cell line
Organisms Homo sapiens; Mus musculus
Characteristics cell type: Embryonic stem cell and NT2 cell line
genotype: Pcl1-3 TKO EV
antibody: RYBP
Growth protocol ESCs that have been maintained in a pluripotent state through culturing with LIF on gelatinised plates. ChIPs include a 10% spike in of human NT2 chromatin that were cultured according to established protocols.
Embryonic stem cells (ESCs) were grown on gelatinised culture dishes in GMEM (sigma) supplemented with 10% ES cell qualified FBS (Millipore), 100 U/mL penicillin (Gibco), 100 U/mL streptomycin (Gibco), 50µM β-mercaptoethanol (sigma), 1:100 Glutamax (Gibco), 1:100 non-essential amino acids (Gibco), 1mM sodium pyruvate (Gibco) and 1:500 homemade leukemia inhibitory factor (LIF). S2 cells were cultured in Schneider media supplemented with 10% Heat inactivated FBS
Extracted molecule genomic DNA
Extraction protocol Embryonic stem cells were washed once with PBS before crosslinking for 10 minutes with PBS containing 1% formaldehyde (Sigma). Crosslinking was quenched with 0.125M Glycine for 5 minutes before two PBS washes. The crosslinked cells were lysed in 6mL of SDS-Lysis buffer (100mM NaCl, 50mM Tris pH8.1, 5mM EDTA pH 8.0, 0.02% NaN3, 0.5% SDS, 2μg/mL Aprotonin, 1μg/mL Leupeptin, 1mM PMSF). Chromatin was pelleted by centrifugation at 1200rpm for 5 minutes at room temperature. The supernatant was then discarded, and the chromatin was resuspended in 3mL of ChIP buffer (2:1 dilution of SDS-Lysis buffer: Triton dilution buffer [100mM Tris pH 8.6, 100mM NaCl, 5mM EDTA pH 8.0, 0.02% NaN3, 5% Triton X-100, 2μg/mL Aprotonin, 1μg/mL Leupeptin, 1mM PMSF]). Chromatin was sheared to approximately 200bp-600bp fragments by sonication on a Branson Sfx150 Sonifier for a total of 4min at 50% amplitude. Sonicated chromatin was incubated overnight with antibody while rotating at 4°C. Following clarification, the chromatin was incubated for 3 hours with 50µL of protein A or G Dyna beads (ThermoFisher) beads. After incubation, the beads were washed three times in Mixed Micelle Buffer (150mM NaCl, 20mM Tris pH 8.1, 5mM EDTA pH 8.0, 5.2% Sucrose, 0.02% NaN3, 1% Triton X-100, 0.2% SDS), twice with Buffer 500 (0.1% Sodium Deoxycholate, 1mM EDTA pH 8.0, 50mM HEPES pH7.5, 1% Triton X-100, 0.02% NaN3), twice with LiCl detergent wash (0.5% Sodium Deoxycholate, 1mM EDTA pH 8.0, 250mM LiCl, 0.5% NP-40, 10mM Tris pH 8.0, 0.02% NaN3) and finally one wash with TE. Immunoprecipitated material was eluted from the beads with Elution buffer (0.1M NaHCO3, 1% SDS) while shaking for 1 hour at 65°C. The supernatant was retained and incubated overnight at 65°C while shaking to reverse the crosslinks. The eluted complexes were then subject to RNase (Thermo Fisher) and Proteinase K (Sigma) treatment prior to DNA clean up (Qiagen Qiaquick PCR Purification Kit). ChIP enrichments were analysed by qPCR using the SYBR Green I detection chemistry (M3003E NEB) on an Applied Biosystems Quant Studio 3 platform.
Following the ChIP experiment, the precipitated DNA was quantified using the Qubit dsDNA High Sensitivity Assay Kit (ThermoFisher Q32854). A Total of 2-10 ng of DNA from each ChIP-Rx experiment was used for library preparation using the NEBNext Ultra II DNA Library Kit for Illumina (E7645) and NEBNext Multiplex Oligos for Illumina (Set#1, NEB #7335). Following adaptor ligation, DNA was PCR amplified for 5-9 cycles, depending on amount of input DNA. DNA purification was then performed using NEBNext Sample Purification Beads (E7767S). The quality of DNA libraries was analysed on a High Senstivity D1000 Screen Tape (Agilent). The resulting libraries were then used for cluster generation and sequencing using an Illumina NextSeq 500 (ID: NB501524), with 75bp read length.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NextSeq 500
Data processing Align reads to reference genome (hg19) using bowite2 and the --very-sensitive-local parameter
Align reads to spike-in genome (mm9 or dm6) using bowtie2 and the --very-sensitive-local parameter
Convert sams to sorted bams using Samtools, and extract headers from both alignments for each of the 2 alignments (reference and spike-in)
Filter read ambiguosly aligned to both genomes using Picards FilterSamReads, mark duplicates using Picards MarkDuplicates and index the filtered, marked bam file with Samtools
The number of spike-in reads was used as a scale factor to generate normalised bigWigs using bamCoverage from the deeptools suite with a bin size of 10
Genome_build: hg19, mm9 & dm6
Supplementary_files_format_and_content: bigwig files of ChIP-Rx spike-in normalised tracks for each sample
Submission date Jun 27, 2019
Last update date Sep 12, 2019
Contact name Craig Monger
Organization name Trinity College Dublin
Department Smurfit Institute of Genetics
Street address College Green
City Dublin 2
State/province Ireland
ZIP/Postal code EIRE
Country Ireland
Platform ID GPL19415
Series (2)
GSE127121 Variant PRC2.1 and PRC2.2 co-operate to direct H3K27 methylations in ESCs
GSE133412 PRC2.1 and PRC2.2 synergize to co-ordinate H3K27 tri-methylation. 
BioSample SAMN12147630
SRA SRX6370186

Supplementary file Size Download File type/resource 58.4 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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