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Sample GSM3660461 Query DataSets for GSM3660461
Status Public on May 15, 2019
Title HiC_2.7kb_clone_replicate1
Sample type SRA
 
Source name HiC_2.7kb_clone
Organism Mus musculus
Characteristics cell line: ESC
clone: 2.7kb teto integrations
Treatment protocol no treatment
Growth protocol Cells were cultured on gelatin-coated culture plates in Dulbecco Modified Eagle’s medium (Sigma) in the presence of 15% foetal calf serum (Eurobio Abcys), 100 µM β-mercaptoethanol, 20 U/ml leukemia inhibitory factor (Miltenyi Biotec, premium grade) in 8% CO2 at 37°C. After insertion of the rTetR-Dam vector (see below), cells were cultured in the presence of 250 µg/mL hygromycin.
Extracted molecule genomic DNA
Extraction protocol see library construction protocol
6x10^6 mESC were harvested and diluted in 1x PBS to final 1x106 cells/ml, then crosslinked with 1% formaldehyde and quenched with 0,125M glycine for 5 min at RT. After two 1x PBS washes, cells pellets were obtained by centrifugation, snap frozen and stored at -80°C. Pellets were thawed on ice and resuspended in 500 ul lysis buffer (10 nM Tris-HCl pH8.0, 10 nM NaCl, 0.2%NP40, 1x Roche protease inhibitors) and left 30 min on ice. Cells were then pelleted by centrifugation (954 x g, 5 min, 4°C ), washed once with 300 ul 1x NEB2 buffer and nuclei were extracted by 1 h incubation at 37°C in 190 ul 0.5%SDS 1xNEB2 buffer. SDS was neutralized by diluting the sample with 400 ul NEB2 buffer and adding 10% Triton X-100. After 15 min of incubation at 37°C, nuclei were pelleted, washed once in PBS and resuspended in 300 ul NEB2 buffer. 400U of MboI (NEB, 25 000 units/ml) were added and incubated at 37°C overnight. The next day, nuclei were pelleted again, resuspended in 200 ul fresh NEB2 buffer and additional 200U of MboI were added for two more hours before heat inactivation at 65°C for 15 min. 43 ul of end-repair mix (1.5 μL of 10 mM dCTP; 1.5 μL of 10mM dGTP; 1.5 μL of 10 mM dTTP; 37.5 μL of 0.4 mM Biotin-11-dATP (Invitrogen) and 1 μL of 50U/μL DNA Polymerase I Large Klenow fragment (NEB)) were added to the nuclear suspension, incubated at 37°C for 45 min and heat inactivated at 65°C for 15 min. The end repair mix was exchanged with 1.2 ml of ligation mix (120μL of 10X T4 DNA Ligase Buffer; 100 μL of 10% Triton X-100; 6 μL of 20 mg/mL BSA; 969μL of H2O) plus 5 ul of T4 ligase (NEB, 2000 units/ml) and ligation was performed at 16°C overnight. Nuclei were reconstituted in 200 ul fresh NEB2 buffer followed by RNA digestion in 0.5 mg/ml RNAse A for 10 min at 37°C. Samples were de-crosslinked with Proteinase K at 65°C overnight and DNA was purified using phenol/chloroform. 2 ug of DNA sample were sonicated using Diagenode Bioruptor Pico. MyOne Streptavidin T1 (Life Technologies # 65601) magnetic beads were used to capture biotinylated DNA followed by A-tailing. Adapter ligation was performed according to NEB Next Ultra DNA Library prep kit instructions. Two independent PCR reactions with multiplex oligos for Illumina sequencing were performed and pooled for the final PCR clean-up by magnetic AMPure bead (Beckman Coulter) purification. The final libraries were eluted in nuclease-free water, QCed by Bioanalyzer and Qubit
 
Library strategy Hi-C
Library source genomic
Library selection other
Instrument model Illumina NextSeq 500
 
Description HiC_2.7kb_pure_clone_replicate1
Data processing Hi-C paired end datasets were processed using HiC-Pro (version 2.7.10). HiC-Pro covers the whole analysis from fastq files to the interaction matrix
Genome_build: mm9
Supplementary_files_format_and_content: iced and binned matrix (10kb). The format of the matrix is the standard from HiC-Pro, namely, 3 columns: start end interactions. It's a sparce matrix annotation, no zero values are reported.
 
Submission date Mar 07, 2019
Last update date Jun 01, 2024
Contact name Luca Giorgetti
Organization name Friedrich Miescher Institute for Biomedical Research
Street address Fabrikstrasse 24
City Basel
ZIP/Postal code 4056
Country Switzerland
 
Platform ID GPL19057
Series (2)
GSE128015 DamC reveals principles of chromatin folding in vivo without crosslinking and ligation [HiC]
GSE128017 DamC reveals principles of chromatin folding in vivo without crosslinking and ligation
Relations
BioSample SAMN11083068
SRA SRX5493739

Supplementary file Size Download File type/resource
GSM3660461_WT_10000_iced.matrix.gz 2.2 Gb (ftp)(http) MATRIX
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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