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| Status |
Public on Aug 11, 2021 |
| Title |
ESC +RNA Ctrl -H3K9me2 repl2 |
| Sample type |
SRA |
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| Source name |
Embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: E14
antibody: H3K9me2 ab1220 lot:GR183500-3
treatment: transfection with RNA Ctrl
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| Treatment protocol |
9 x 105 ESC were plated in gelatin-coated 10cm culture dish and transfected with 42 μg pRNA, or RNA-control using Lipofectamine MessengerMAX reagent (Invitrogen) in Opti-MEM GlutaMAX (Life Technologies) reduced-serum medium for 48h
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| Growth protocol |
E14 mouse embryonic stem cells were cultured in either 2i media composed of DMEM-F12 and Neurobasal medium (1:1, Life Technologies), supplemented with 1× N2/B27 (Life Technologies), 1× penicillin/streptomycin/l-glutamine (Life Technologies), 50 μM β-mercaptoethanol (Life Technologies), recombinant leukemia inhibitory factor, LIF (Polygene, 1,000 U/ml) and MEK and GSK3β inhibitors, 2i (Sigma CHIR99021 and PD0325901, 3 and 1 μM, respectively). ESCs were seeded at a density of 50,000 cells/cm2 in culture dishes (Corning® CellBIND® surface) coated with 0.1% gelatin without feeder layer. Propagation of cells was carried out every 2 days using enzymatic cell dissociation.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
1% formaldehyde was added to cultured cells to cross-link proteins to DNA. Isolated nuclei were then lysed with lysis buffer (50mM Tris-HCl, pH 8.1, 10 mM EDTA, pH 8, 1% SDS, 1X protease inhibitor cOmplete EDTA-free cocktail, Roche). Nuclei were sonicated using a Bioruptor ultrasonic cell disruptor (Diagenode) to shear genomic DNA to an average fragment size of 200 bp. 20 μg of chromatin was diluted to a total volume of 500 μl with ChIP buffer (16.7 mM Tris–HCl, pH 8.1, 167 mM NaCl, 1.2 mM EDTA, 0.01% SDS, 1.1% Triton X-100) and incubated overnight with the ChIP-grade antibodies against H3K9me2 and H3K9me3. After washing, bound chromatin was eluted with the elution buffer (1% SDS, 100 mM NaHCO3). Upon proteinase K digestion (50°C for 3 h) and reversion of cross-linking (65°C, overnight), DNA was purified with phenol/chloroform, ethanol precipitated and quantified by qPCR.
The Nugen Ovation Ultra Low Library Systems (Nugen, Inc, California, USA) was used in the following steps. ChIP samples (1 ng) was end-repaired and polyadenylated before the ligation of Illumina compatible adapters. The adapters contain the index for multiplexing. The quality and quantity of the enriched libraries were validated using Qubit® (1.0) Fluorometer and the Bioanalyzer 2100 (Agilent, Waldbronn, Germany). The libraries were normalized to 10nM in Tris-Cl 10 mM, pH8.5 with 0.1% Tween 20. The TruSeq SR Cluster Kit v4-cBot-HS (Illumina, Inc, California, USA) was used for cluster generation using 8 pM of pooled normalized libraries on the cBOT. Sequencing was performed on the Illumina HiSeq 2500 single end 126 bp using the TruSeq SBS Kit v4-HS (Illumina, Inc, California, USA).
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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 |
Fastaq files were aligned to the mm10 genome assembly using Bowtie2 (version 2.3.4.3) with default parameters
The bam files were analyzed using the damidseq pipeline script from the Brand group (http://owenjm.github.io/damidseq_pipeline) (Marshall and Brand, 2015). The pipeline bins the mapped reads into GATC-fragments according to GATC-sites indicated by a gff file for the GRCm38 mouse genome (already provided by the authors of the pipeline on the website above) and normalizes reads against the Dam control, in our case the H2B-Dam sample. The pipeline gives a bedgraph file with the log2 ratio of the m6A between H2B-Dam-NoLS and the H2B-Dam only. The bedgraph files were processed with the find_peaks software associated with the pipeline (https://github.com/owenjm/find_peaks) adjusting the values of the FDR and the minimum quantile (FDR <0.01 and min_quant 0.70). Only the significative peaks common to both replicates were considered as NADs for further analysis. The intersection of identified NADs and previously published lamina associated domains (LADs, Peric-Hupkes et al., 2010) results in the classification of NADonly, NAD/LAD, and LAD.
for ChIP seq data,read counts were computed and normalized using “bamCoverage” from deepTools (version 3.2.1) using a bin size of 50bp.
for RNA SEQ, reads were aligned to the reference genome (ensembl version 82) with Subread (i.e. subjunc, version 1.4.6-p4; 50) allowing up to 16 alignments per read (options: –trim5 10 –trim3 15 -n 20 -m 5 -B 16 -H –allJunctions). Count tables were generated with Rcount 51 with an allocation distance of 100 bp for calculating the weights of the reads with multiple alignments, considering the strand information, and a minimal number of 5 hits. For each gene, exon coverage was calculated using a custom pipeline and then normalized in reads per kilobase per million (RPKM) (Mortazavi et al., 2008), the method of quantifying gene expression from RNA sequencing data by normalizing for total read length and the number of sequencing reads.
Genome_build: mm10
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| Submission date |
Mar 31, 2021 |
| Last update date |
Aug 11, 2021 |
| Contact name |
Raffaella Santoro |
| E-mail(s) |
raffaella.santoro@dmmd.uzh.ch
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| Phone |
+41 44 635 54 75
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| Organization name |
University of Zurich
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| Department |
Dep. of Molecular Mechanisms of Disease
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| Street address |
Winterthurerstrasse 190
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| City |
Zurich |
| ZIP/Postal code |
8057 |
| Country |
Switzerland |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE150822 |
Identification of nucleolar associated domains (NADS) |
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| Relations |
| BioSample |
SAMN18577167 |
| SRA |
SRX10489533 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5221280_ESC_RNAControl_K9me2_repl2_R1.bw |
63.6 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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