NCBI Logo
GEO Logo
   NCBI > GEO > Accession DisplayHelp Not logged in | LoginHelp
GEO help: Mouse over screen elements for information.
          Go
Sample GSM1718021 Query DataSets for GSM1718021
Status Public on Aug 12, 2015
Title Human sample1: Hi-C in-solution ligation
Sample type SRA
 
Source name H9 (WA09; WiCell) human ESCs
Organism Homo sapiens
Characteristics cell type: ESC
cell line: H9 (wa09; wicell)
developmental stage: embryo
Growth protocol H9 (WA09; WiCell) human ESCs were maintained using Pluripro fully-defined media and matrix (Cell Guidance Systems). Mouse foetal livers were dissected from C57BL/6 mouse embryos at day 14.5 (E14.5) of development.
Extracted molecule genomic DNA
Extraction protocol Approximately 50 million human ESCs (at passage 56) were harvested with Accutase (Life Technologies), suspended in Pluripro media and directly processed for fixation. Mouse foetal liver cells were suspended in DMEM (Life Technologies) supplemented with 10% FBS, filtered through a cell strainer (70 µm) and directly fixed by addition of formaldehyde.
Thirty to 50 million cells were fixed in 2% formaldehyde for 10 min, quenched with 0.125 M glycine, spun down (400 x g, 5 min) and washed once with PBS. The cells were incubated in 50 ml permeabilisation buffer (10 mM Tris-HCl pH 8, 10 mM NaCl, 0.2% Igepal CA-630, Complete EDTA-free protease inhibitor cocktail [Roche]) for 30 min on ice with occasional agitation, spun down (650 x g, 5 min, 4˚C), and the cell pellets were resuspended in 358 µl of 1.25 x NEBuffer2 (NEB) per 5 million cell aliquot. Eleven µl of 10% SDS was added to each aliquot, followed by an incubation at 37˚C for 60 min with continuous agitation (950 rpm). To quench the SDS, 75 µl of 10% Triton X-100 was then added per aliquot, followed by an incubation at 37˚C for 60 min with continuous agitation (950 rpm). To digest chromatin, 1500 U of Hind III (NEB) was added per aliquot and incubated at 37˚C overnight with continuous agitation (950 rpm). After digestion, restriction sites were filled in with Klenow (NEB) in the presence of biotin-14-dATP (Life Technologies), dCTP, dGTP and dTTP (all 30 µM) for 60 min at 37˚C. For in-solution ligation, 86 µl of 10% SDS was added per aliquot and incubated at 65˚C for 30 min with continuous agitation (950 rpm), followed by addition of 7.61 ml of ligation mix (745 µl of 10% Triton X-100, 820 µl of 10 x T4 DNA ligase reaction buffer [NEB], 82 µl of 10 mg/ml BSA [NEB] and 5.965 ml water) per aliquot and incubation at 37˚C for 60 min with occasional agitation. For in-nucleus ligation, 7.61 ml of ligation mix (820 µl of 10 x T4 DNA ligase reaction buffer [NEB], 82 µl of 10 mg/ml BSA [NEB] and 6.71 ml water) was added per aliquot (compared to the in-solution ligation, SDS addition and incubation at 65˚C were omitted). For the ligation reaction (both in-solution and in-nucleus variants), 50 µl of 1 U/µl T4 DNA ligase (Life Technologies) was added per aliquot, followed by incubation at 16˚C for 4 hrs. The cross-links were reversed by adding 60 µl of 10 mg/ml proteinase K (Roche) per aliquot and incubating at 65˚C overnight. After overnight incubation, another 60 µl of proteinase K per aliquot was added, followed by incubation at 65˚C for an additional two hours. RNA was removed by adding 12.5 µl of 10 mg/ml RNase A (Roche) per aliquot and incubating at 37˚C for 60 min. DNA was isolated by a phenol (Sigma) extraction, followed by a phenol/chloroform/isoamylalcohol (Sigma) extraction and standard ethanol precipitation. The precipitated DNA was washed three times with 70% ethanol, and dissolved in 25 µl TE per aliquot. Subsequently all aliquots were pooled and the Hi-C DNA was quantified (Quant-iT Pico Green, Life Technologies). Biotin was removed from non-ligated restriction fragment ends by incubating 30 to 40 µg of Hi-C library DNA with T4 DNA polymerase (NEB) for 4 hours at 20˚C in the presence of dATP. After DNA purification (Qiagen PCR purification kit) and sonication (Covaris E220), the sonicated DNA was end-repaired with T4 DNA polymerase, T4 DNA polynucleotide kinase, Klenow (all NEB) and dNTPs in 1 x T4 DNA ligase reaction buffer (NEB). Double size selection of DNA was performed using AMPure XP beads (Beckman Coulter), before dATP-addition with Klenow exo- (NEB). Biotin-marked ligation products were isolated with MyOne Streptavidin C1 Dynabeads (Life Technologies) in binding buffer (5 mM Tris pH8, 0.5 mM EDTA, 1 M NaCl) for 30 min at room temperature, followed by two washes in binding buffer, and one wash in 1 x T4 DNA ligase reaction buffer (NEB). PE adapters (Illumina) were ligated onto Hi-C ligation products bound to streptavidin beads for 2 hours at room temperature (T4 DNA ligase in 1 x T4 DNA ligase reaction buffer [NEB], slowly rotating). After washes in wash buffer (5 mM Tris, 0.5 mM EDTA, 1 M NaCl, 0.05% Tween-20) and binding buffer, the DNA-bound beads were resuspended in NEBuffer 2. Bead-bound Hi-C DNA was amplified with 12 PCR amplification cycles using PE PCR 1.0 and PE PCR 2.0 primers (Illumina). The concentration and size distribution of Hi-C library DNA after PCR amplification was determined by Bioanalyzer profiles (Agilent Technologies) and quantitative PCR, and the Hi-C libraries were paired-end sequenced on Illumina Hi-Seq1000 or MiSeq platforms.
 
Library strategy OTHER
Library source genomic
Library selection other
Instrument model Illumina HiSeq 1000
 
Data processing Library strategy: Hi-C
Hi-C analysis pipeline HiCUP v0.4.2 (running bowtie2 v2.2.3 for mixed human-mouse samples or bowtie v0.12.9 for other samples). HiCUP homepage: www.bioinformatics.babraham.ac.uk/projects/hicup
Hi-C normalisation used Hicpipe v0.93 (Hicpipe homepage: http://compgenomics.weizmann.ac.il/tanay/?page_id=283 )
Hi-C normalisation also used Homer (Homer homepage: http://homer.salk.edu/homer )
Genome_build: hg19 or mm9
Supplementary_files_format_and_content: The files with extension ".raw" provide the positions of interacting Hi-C read pairs (di-tags). The header line in each file describes each column.
 
Submission date Jun 23, 2015
Last update date Aug 12, 2015
Contact name Steven William Wingett
E-mail steven.wingett@babraham.ac.uk
Organization name Babraham Institute
Department Bioinformatics
Street address Babraham Research Park
City Cambridge
State/province Cambs
ZIP/Postal code CB22 3AT
Country United Kingdom
 
Platform ID GPL15433
Series (1)
GSE70181 Comparison of Hi-C results using in-solution versus in-nucleus ligation
Relations
BioSample SAMN03787383
SRA SRX1070631

Supplementary file Size Download File type/resource
GSM1718021_human1_ISL_CAGATC_L001_R.raw.txt.gz 117.2 Mb (ftp)(http) TXT
Raw data provided as supplementary file
Processed data provided as supplementary file

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