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| Status |
Public on Mar 07, 2017 |
| Title |
H3K27me3 ChIP (haploid) |
| Sample type |
SRA |
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| Source name |
mESCs
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| Organism |
Mus musculus |
| Characteristics |
cell type: haploid mESCs derived from strain 129/Ola
chip antibody: antibody: millipore (07-449)
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| Growth protocol |
All mouse embryonic stem cells (mESCs) were cultured on 0.2 % gelatin in 2i media (NDiff B27 base medium, Stem Cell Sciences Ltd, catalogue no: SCS-SF-NB-02, supplemented with 1 uM PD0325901, 3 uM CHIR99021 and 20 ng/ml LIF). Haploid mESCs were sorted every 4 passages to enrich for haploid cells as previously described in Leeb M & Wutz A, Nature, 2011
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP-seq samples were prepared as described in Morey et al, 2015. Briefly, 5 million mESCs were removed from the dish using accutase and resuspended in 2i media. mESCs were then fixed in 1 % formaldehyde for 10 min before adding 0.0125 M glycine for 5 min to quench the reaction. Chromatin was then immunoprecipitated with 5 ug of antibody
Libraries were prepared according to Illumina's instructions accompanying the DNA Sample Kit (Part# 0801-0303). Briefly, DNA was end-repaired using a combination of T4 DNA polymerase, E. coli DNA Pol I large fragment (Klenow polymerase) and T4 polynucleotide kinase. The blunt, phosphorylated ends were treated with Klenow fragment (32 to 52 exo minus) and dATP to yield a protruding 3- 'A' base for ligation of Illumina's adapters which have a single 'T' base overhang at the 3’ end. After adapter ligation DNA was PCR amplified with Illumina primers for 15 cycles and library fragments of ~250 bp (insert plus adaptor and PCR primer sequences) were band isolated from an agarose gel. The purified DNA was captured on an Illumina flow cell for cluster generation. Libraries were sequenced on the Genome Analyzer following the manufacturer's protocols.
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Basecalls performed using Phred quality score (Q score)
ChIP-seq reads were aligend to the GRCm38/mm10 mouse genome reference using Bowtie 2 v2.1.050 and filtered to retain reads with mapping quality >30.
ChIP-seq peaks were called using MACS2 v2.1.0.2015073157 with a minimum FDR cutoff of 0.01 (-q 0.01), except for broad features (H3K27me3, H3K36me3 and H3K9me3) where a cutoff of 0.05 was used (-q 0.05).
ChIP-seq peaks were filtered to remove those not corresponding to the canonical chromosomes.
RNA-seq transcript abundance estimates for all annotated mouse transcripts (Ensembl v71, GRCm38/mm10) and ERCC spike-in sequences were obtained using Kallisto v0.42.4 (arXiv:1505.02710).
RNA-seq gene-level abundances were summarized from transcript-level estimates using the tximport R package.
Population Hi-C reads were processed using the HiCUP software package (http://www.bioinformatics.babraham.ac.uk/projects/hicup/overview/). Processed reads were aligned to the GRCm38/mm10 mouse genome reference using Bowtie 2 and filtered to retain reads that formed a valid Hi-C contact junction between two RE1 resriction sites.
Single cell Hi-C reads were initially processed using the NucProcessing software package (available upon request). Processed reads were aligned to the GRCm38/mm10 mouse genome reference using Bowtie 2 and filtered to retain reads that formed a valid Hi-C contact junction between two RE1 resriction sites. Output files were further processed to perform extra, single-cell specific processing and cleanup. These remove any pairs that represent only a single observation of a specific RE1-RE1 ligation junction after PCR amplification, while at least two separate, albeit sometimes identical, molecules must be paired-end sequenced to confirm a ligation junction. Next the sequence pairs were filtered to remove those with promiscuous ends: where the RE1 fragment at either end was involved in more than one ligation event. Finally, the redundancy in amplified RE1-RE1 ligation events was removed to create a single list of paired RE1 fragment ends.
Genome_build: mm10
Supplementary_files_format_and_content: bed files (.bed) are tab-delimited text files containing chromosomal coordinates of all ChIP-seq peaks (chromosome name, start, end).
Supplementary_files_format_and_content: tsv files (.tsv) contain gene-level abundance estimates for all genes.
Supplementary_files_format_and_content: Single cell Hi-C txt file (.txt) is a tab-separated text format containing the Hi-C contacts. After the header line, each individual contact is represented on a line consisting of chromosome_1 seq_pos_1 chromosome_2seq_pos_2
Supplementary_files_format_and_content: Population Hi-C txt file (.txt) is a tab-separated text format containing all the chromosomal coordinates for Hi-C contacts and the number observed (chromosome_A start position_A end position_A chromosome_B start position_B end position_B number observed)
Supplementary_files_format_and_content: Single cell genome structure pdb file (.pdb) is protein data bank format. Atom type N is used to indicate restrained particles. Atom type C is used to represent unrestrained backbone particles. Residue number corresponds to the particle and increases every 100 kb. Chain letter represents the chromosome. The last column represents the particle sequence position.
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| Submission date |
Apr 14, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Andre J Faure |
| E-mail(s) |
andre.faure@crg.eu
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| Organization name |
Centre for Genomic Regulation (CRG)
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| Department |
EMBL-CRG Systems Biology Unit
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| Street address |
Dr. Aiguader 88
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| City |
Barcelona |
| ZIP/Postal code |
08003 |
| Country |
Spain |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE80280 |
3D structures of individual mammalian genomes reveal principles of nuclear organization |
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| Relations |
| BioSample |
SAMN04855978 |
| SRA |
SRX1704885 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2123548_H3K27me3_hapmESC_peaks.bed.gz |
575.7 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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