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| Status |
Public on Mar 17, 2020 |
| Title |
mES_Mll2KO_100nM5dAza_mRRBS_rep1 |
| Sample type |
SRA |
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| Source name |
embryonic stem cell
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| Organism |
Mus musculus |
| Characteristics |
cell type: embryonic stem cell
cell line: v6.5
drug treatment: 100nM5dAza
genotype/variation: Mll2KO
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| Treatment protocol |
differentiation protocol: 1500 ESCs were cultured in 25 µL of EB differentiation medium on the lid of 150-mm culture plates for 6 d. EB differentiation medium was composed of DMEM supplemented with 15% FBS (Gemini Bio-Products), 1× GlutaMAX (Gibco), 1× MEM nonessential amino acids (Gibco), 1× β-mercaptoethanol (Gibco), and 1× penicillin–streptomycin (Gibco).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
RNA was extracted using RNeasy mini kit (Qiagen) and RNAse free DNaseI (Sigma) was used to eliminate DNA contamination.
For ChIPseq, ESCs were fixed with 1% formaldehyde for 10 minutes. After quenching and cell lysis, chromatin were sonicated using a E220 focused-ultrasonicator (Covaris). Sheared chromatin, 5 µg antibody, and 50 µl protein A/G beads (Santa Cruz) were used for each immunoprecipitation. Immunoprecipitated DNA were purified after washing, eluting, and reverse-crosslinking and submitted for library preparation.
For PROseq, nuclei were isolated by Dounce homogenizer with loose pestle. Nuclei were subjected to nuclear run-on in the presence of biotin-11-ATP/GTP/CTP/UTP and Drosophila S2 spike-in nuclei. Biotinylated nascent RNA was purified by Dynabeads M-280 streptavidin.
For mRRBS, genomic DNA was digested with MspI (New England BioLabs) before size selection of 100- to 250-bp fragments with solid-phase reversible immobilization beads (MagBio Genomics). DNA was bisulfite converted with the EZ DNA Methylation-Lightning Kit.
ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific. 10 ng of DNA was used as starting material for input and ip samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected at 250- 450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit. RNA-seq libraries were prepared using the Illumina TruSeq Stranded Total RNA Preparation Kit with Ribo-Depletion. Input RNA quality was validated using the Agilent RNA 6000 Nano Kit. 200ng-1 µg of total RNA was used as starting material. Libraries were validated using the Agilent DNA 1000 Kit
ChIP-seq libraries were prepared using the KAPA HTP Library Preparation Kit complemented with NEXTflex DNA Barcodes from Bioo Scientific. 10 ng of DNA was used as starting material for input and ip samples. Libraries were amplified using 13 cycles on the thermocycler. Post amplification libraries were size selected at 250- 450bp in length using Agencourt AMPure XP beads from Beckman Coulter. Libraries were validated using the Agilent High Sensitivity DNA Kit. RNA-seq libraries were prepared using the Illumina TruSeq Stranded Total RNA Preparation Kit with Ribo-Depletion. Input RNA quality was validated using the Agilent RNA 6000 Nano Kit. 200ng-1 µg of total RNA was used as starting material. Libraries were validated using the Agilent DNA 1000 Kit
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
fragmented genomic DNA
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| Data processing |
Basecalls were performed using bcl2fastq.
Both ChIP-seq and RNA-seq reads were trimmed from 3' end until the final base had a quality score > 30, using Trimmomatic v0.33, discarding reads left with < 20 bp
ChIP-seq reads were aligned to UCSC mm9 using Bowtie version 0.12.9. Only uniquely mapping reads with at most two mismatches were retained.
RNA-seq reads were aligned to UCSC mm9 using Tophat version 2.0.9. Only uniquely mapping reads with at most two mismatches were retained.
To create ChIP-seq coverage plots, the locations of the mapped ChIP-seq reads were extended to 150 bp to represent sequenced fragments, renormalized (to reads per million, rpm) and reformatted in the bigWig file format.
For PRO-seq analyses, PRO-seq raw data were trimmed by cutadapt 1.14 and Trimmomatic 0.33. Each sample was next mapped to the mouse and fruit fly genome assemblies (mm9 and dm3, respectively) using Bowtie. The number of fly reads on each experiment was used to normalize the corresponding genome-wide strand-specific profiles.
For CRISPR screen sequencing, reads were aligned to the sgRNAs using Bowtie1. For CRISPR screen counts, the enrichment of individual guides was calculated as the log2 ratios ‘Sorted cells’ and ‘Total population’, with each sgRNA with a log2FC > 1 considered as enriched.
For mRRBS, well-observed CpG positions were obtained by performing an analysis of variance (ANOVA)–like test for differential methylation with the DSS v2.26.0 R/Bioconductor package and quantified using the SeqMonk platform (v1.40.1) with the bisulfite feature methylation pipeline.
Genome_build: mm9
Supplementary_files_format_and_content: bigWig files were generated using the genomicRanges R package. Score represents the normalized coverage of RNA or DNA fragments at a given genomic coordinate.
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| Submission date |
Feb 25, 2020 |
| Last update date |
Mar 17, 2020 |
| Contact name |
Ali Shilatifard |
| E-mail(s) |
ash@northwestern.edu
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| Organization name |
Northwestern University Feinberg School of Medicine
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| Department |
Department of Biochemistry and Molecular Genetics
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| Lab |
Shilatifard Lab
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| Street address |
320 E Superior St
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| City |
Chicago |
| State/province |
IL |
| ZIP/Postal code |
60611 |
| Country |
USA |
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| Platform ID |
GPL19057 |
| Series (1) |
| GSE129037 |
Uncoupling histone H3K4 trimethylation from developmental gene expression via an epigenetic equilibrium of Polycomb, COMPASS and DNA methylation |
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| Relations |
| BioSample |
SAMN14206813 |
| SRA |
SRX7800442 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4339124_mES_Mll2KO_100nM5dAza_mRRBS_rep1.cov.gz |
75.2 Mb |
(ftp)(http) |
COV |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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