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| Status |
Public on Jan 25, 2023 |
| Title |
Mll3KOMll4flox_D4_MLL4 |
| Sample type |
SRA |
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| Source name |
Embryoid bodies (EBs)
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| Organism |
Mus musculus |
| Characteristics |
genotype: Mll3-/-;Mll4flox/flox
cell type: ES-derived EBs
stage of differentiation: D4
antibodies: MLL4 (homemade, #3)
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Cells were cross-linked with 1% formaldehyde for 10 min and quenched by 125 mM glycine for 10 min. 30 million fixed cells were swelled in cold buffer containing 5mM PIPES (pH 8.0), 85mM KCl, 1% NP-40 and protease inhibitors for 20 min, and centrifuged at 2,000 g for 5 min at 4°C. Nuclei were resuspended with 3 mL TE buffer (pH 8.0), and subjected to sonication. Sheared chromatin was clarified by centrifugation at 4,000 g for 10 min at 4°C. The supernatant was transferred to a new tube and further supplemented with 150mM NaCl, 0.1% SDS, 1% Triton-X100 and protease inhibitors. 2% of the total material was set aside as input and 20 ng of spike-in chromatin (Active Motif, #53083) was added to each ChIP material. For each ChIP material, 2 - 8 µg of target antibodies and 1.5 μg of spike-in antibody (anti-H2Av, Active Motif, #61686) pre-bound with protein A Dynabeads (ThermoFisher) were added and incubated on a rotator at 4°C overnight. Beads were then collected on a magnetic rack and washed twice with 1 mL cold RIPA buffer, once with 1 mL cold RIPA buffer containing 500mM NaCl, twice with 1 mL cold LiCl buffer and once with 1 mL TE buffer. Finally, beads were eluted with 100 μL buffer containing 0.1M NaHCO3, 1% SDS, and 100 μg Proteinase K at 65°C overnight. Samples were then purified using QIAquick PCR purification kit (Qiagen) and eluted in 50 μL 10mM Tris-HCl.
Libraries were prepared according to NEB (Illumina kit) instructions. DNA were used to construct libraries using NEBNext® Ultra™ II DNA Library Prep kit with AMPure XP magnetic beads (Beckman Coulter).
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| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 3000 |
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| Data processing |
Basecalls performed using Bcl2fastq.
ChIP-seq reads were aligned to the mm9 genome assembly using bowtie2.
Peaks were called using SICER with following configuration - For ChIP-Seq of histone modifications: Windows (200), Gaps (200), FDR (1e-3); for ChIP-Seq of MLL4: Windows (50), Gaps (50), FDR (1e-10)
Single-end reads of RNA-seq are aligned to the mm9 genome assembly using STAR.
SICER v1.1 was used to calculate the reads per kilo base per million (RPKM) on exonic regions as expression measurements for each gene.
Genome_build: mm9
Supplementary_files_format_and_content: wig files were generated using in-house script and the scores represent RPKM; tab-delimited text files include FPKM values for each gene.
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| Submission date |
Jul 15, 2020 |
| Last update date |
Jan 25, 2023 |
| Contact name |
Kai Ge |
| E-mail(s) |
kai.ge@nih.gov
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| Phone |
301-451-1998
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| Organization name |
NIH
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| Department |
NIDDK
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| Street address |
10 Center Dr Rm 8N307
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| City |
Bethesda |
| State/province |
MD |
| ZIP/Postal code |
20892 |
| Country |
USA |
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| Platform ID |
GPL21493 |
| Series (1) |
| GSE154475 |
MLL3/MLL4 methyltransferase activities control early embryonic development and ESC differentiation in a lineage-selective manner |
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| Relations |
| BioSample |
SAMN15543106 |
| SRA |
SRX8736760 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4671860_Mll3KOMll4flox_D4_MLL4_sorted-W50-G50-FDR1e-10-islandfiltered-normalized.wig.gz |
1.4 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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