GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
Sample GSM5627349 Query DataSets for GSM5627349
Status Public on May 23, 2022
Title TetR-PCGF2_ES_rep1
Sample type SRA
Source name Mouse embryonic stem cells, CaptureC
Organism Mus musculus
Characteristics cell line: TetR-PCGF2
genotype: chr8:TetO-BAC:RP11419E6
strain: E14
replicate: 1
treatment agent: none
treatment time point: none
Treatment protocol To induce conditional removal of Med13 and Med13l in MED13/13lfl ESC line or Pcgf2 in Pcgf2fl ESC line, cells were treated with 800nM 4-hydroxytamoxifen (TAM) for 96 hours or 72 hours, respectively.
Growth protocol Mouse embryonic stem cells were grown on gelatin-coated plates at 37°C and 5% CO2, in Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 15% fetal bovine serum (Labtech), 2 mM L-glutamine (Life Technologies), 1x penicillin-streptomycin solution (Life Technologies), 1x non-essential amino acids (Life Technologies), 0.5 mM beta-mercaptoethanol (Life Technologies), and 10 ng/mL leukemia inhibitory factor.
Extracted molecule genomic DNA
Extraction protocol CaptureC libraries were prepared as described previously (Hughes et al., Nature Genetics 2014). Briefly, 10 Million mouse ES cells were trypsinized, collected in 50ml falcon tubes in 9.3ml media and crosslinked with 1.25ml 16% formaldehyde for 10min at room temperature. Cells were quenched with 1.5M glycine, washed with PBS and lysed for 20 minutes at 4oC lysis buffer (10mM Tris pH 8, 10mM NaCl, 0.2% NP-40, supplemented with complete proteinase inhibitors) prior to snap freezing in 1ml lysis buffer at -80oC.
Lysates were then thawed on ice, pelleted and resuspended in 650µl 1x DpnII buffer (NEB). Three 1.5ml tubes with 200µl lysate each were treated in parallel with SDS (0.28% final concentration, 1hr, 37 oC, interval shaking 500rpm, 30sec on/30sec off), quenched with trypsin (1.67% final concentration, 1hr, 37 oC, interval shaking 500rpm, 30sec on/30sec off) and subjected to a 24 hour digestion with 3x10µl DpnII (homemade, 37 oC, interval shaking 500rpm, 30sec on/30sec off). Each chromatin aliquot was independently ligated with 8 µl T4 Ligase (240 U) in a volume of 1440µl (20 hours, 16oC). Following this, the nuclei containing ligated chromatin were pelleted to remove any non-nuclear chromatin, reverse-crosslinked and the ligated DNA was phenol-chloroform purified. The sample was resupended in 300µl water and sonicated 13x (Pico Bioruptor, 30sec on, 30sec off) or until a fragment size of approximately 200bp was reached. Fragments were size selected using AmpureX beads (Beckman Coulter: A63881, selection ratios: 0.85x / 0.4x). 2x 1-5µg of DNA were adaptor ligated and indexed using the NEBNext DNA library Prep Reagent Set (New England Biolabs: E6040S/L) and NEBNext Multiplex Oligos for Illumina Primer sets 1 (New England: E7335S/L) and 2 (New England: E7500S/L). The libraries were amplified 7x using Herculase II Fusion Polymerase kit (Agilent: 600677). Libraries were hybridized in teh following way: Probes were designed using the online tool by the Hughes lab (CapSequm: to be either 120bp (Sox2) or 70bp long each, two probes for each promoter of interest. The probes were pooled at a 2.9nM each and the samples were multiplexed by mass prior to hybridization. Hybridization was carried out using the Nimblegen SeqCap system (Roche, Nimblegen SeqCap EZ HE-oligo kit A # 06777287001, Nimblegen SeqCap EZ HE-oligo kit B # 06777317001, Nimblegen SeqCap EZ Accessory kit v2 # 07145594001, Nimblegen SeqCap EZ Hybridisation and wash kit # 05634261001) according to Roche protocol for 72 hours followed by a 24 hours hybridization (double Capture). The captured libraries were sequenced on Illumina NextSeq.
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina NextSeq 500
Data processing For CaptureC, paired-end reads were aligned and filtered for HiC artefacts using HiCUP (Wingett S, et al. (2015)) and Bowtie2 (Langmead and Salzberg, 2012) with fragment filter set to 100-800bp. Read counts of reads aligning to captured gene promoters and interaction scores (=significant interactions) were then called by CHiCAGO (Cairns et al., 2016).
For visualisation of CaptureC data weighted pooled read counts from chicago data files were normalized to total read count aligning to captured gene promoters in the sample and further to the number of promoters in the respective capture experiment. Bigwig files were generated from these normalized read counts.
For comparative boxplot analysis interactions called by chicago (score >= 5) across all samples were aggregated and interactions with a distance of less than 4 DpnII fragments were merged to a single interaction peak. For each interaction peak we then quantified mean normalized read count and chicago score of all overlapping DpnII fragments.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig files showing the genome coverage of normalized read counts for each sample and each promoter were generated from chicago data object.
Submission date Oct 14, 2021
Last update date May 23, 2022
Contact name Emilia Dimitrova
Organization name University of Oxford
Department Biochemistry
Lab Rob Klose
Street address South Parks Road
City Oxford
ZIP/Postal code OX1 3QU
Country United Kingdom
Platform ID GPL19057
Series (2)
GSE185926 Distinct roles for CDK-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction (CaptureC)
GSE185930 Distinct roles of CKM-Mediator in controlling Polycomb-dependent chromosomal interactions and priming genes for induction
BioSample SAMN22306997
SRA SRX12622620

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

| NLM | NIH | GEO Help | Disclaimer | Accessibility |
NCBI Home NCBI Search NCBI SiteMap