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| Status |
Public on Feb 05, 2024 |
| Title |
4C-seq_Peg13_DMR_ES_BJ |
| Sample type |
SRA |
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| Source name |
ES_BJE2
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| Organism |
Mus musculus |
| Characteristics |
cell line: ES_BJE2
cell type: ES cells
genotype: C57BL/6 x JF1
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| Growth protocol |
ES cell lines were maintained in gelatin-coated dishes with ESGRO complete plus medium (Millipore, SF001-500P) containing LIF, BMP4, and a GSK3-β inhibitor. ES-derived neural precursor cells were generated using in vitro corticogenesis as described in Montibus et al 2021 (PMID: 32405722).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
4C Template Preparation was carried out as previously described with some modifications (Harmen J G van de Werken et al,). Briefly, 1x107 cell suspensions were cross-linked with formaldehyde (final concentration 2%) for 10 min. After cell lysis and permeabilization by SDS and Triton X-100 treatments, samples were digested with 600U DpnII at 37ºC in 1X NEBuffer DpnII (4 h with 200U, overnight with 200U and 4 h with 200U). The restriction enzyme was inactivated with SDS and Triton X-100. The first ligation was performed overnight at 18°C in a large volume, 7.2 ml of 1X ligase buffer and with 50U T4 DNA Ligase. The cross-linking was reversed with 600ug Proteinase K at 65°C overnight. After phenol/chloroform purification, the DNA was digested with 50U of NlaIII overnight at 37°C. After phenol/chloroform purification, a second ligation was performed overnight at 18°C in 14ml of 1X ligase buffer and with 100U of T4 DNA Ligase. The 4C Template was concentrated with an ethanol precipitation and then purified using the DNA Clean & Concentrator Zymo-25 kit.
To produce a 4C-seq library, 3,2 µg of 4C template was amplified into 16 PCR reactions with viewpoint specific sequencing primers and 56 U of Expand Long Template Polymerase. PCR reactions were pooled and purified using High Pure PCR Product Purification Kit. 4C-seq library of different viewpoints were combined before sequencing on an HiSeq 4000 or NovaSeq 6000 instrument (Illumina) by IntegraGen SA.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
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| Data processing |
Due to the primer design, paired end reads were used to determine the viewpoint allele and the interacting sequence. To do this, only the expected sequence, corresponding to the viewpoint of the informative reads, was mapped to the mm39 hybrid genome using bowtie2 (--trim5 10 --trim3 “viewpoint specific” --local --very-sensitive-local). Only alignments mapped to the viewpoint coordinates and with a minimal quality were conserved to determine the origin allelic of the viewpoint in the reads (samtools view -q 10 [ viewpoint coordinates]). To determine the sequence in interaction, the expected sequence with DpnII restriction site was mapped to the mm39 masked genome using bowtie2 (--trim5 “viewpoint specific” --trim3 “viewpoint specific” --local --very-sensitive-local). these alignments were filtered for mapping quality (samtools view -q 10 ) and were split according to the origin allelic of the viewpoint. To construct the allelic interactome, the alignments were processed the FourCSeq Bioconductor package to count the reads mapped exactly to the end of a DpnII fragment and to generate a smoothed rpm normalized coverage.
Assembly: mm39
Supplementary files format and content: bigwig, allelic coverage
Library strategy: 4C-seq
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| Submission date |
Sep 27, 2023 |
| Last update date |
Feb 05, 2024 |
| Contact name |
franck court |
| Organization name |
GReD CNRS
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| Street address |
28 Place Henri Dunant
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| City |
Clermont-Ferrand |
| ZIP/Postal code |
63000 |
| Country |
France |
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| Platform ID |
GPL21103 |
| Series (2) |
| GSE244143 |
Biallelic non-productive enhancer-promoter interaction precedes imprinted expression of KcnK9 during mouse neural commitment [4C-seq] |
| GSE244147 |
Biallelic non-productive enhancer-promoter interaction precedes imprinted expression of KcnK9 during mouse neural commitment |
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| Relations |
| BioSample |
SAMN37560789 |
| SRA |
SRX21902964 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7807809_rpm_counts_5_Peg13_MAT_ES_1.bw |
941.3 Kb |
(ftp)(http) |
BW |
| GSM7807809_rpm_counts_5_Peg13_PAT_ES_1.bw |
1.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
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