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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Feb 02, 2017 |
| Title |
Mll2_Mll2KO_MLL2_deltaCXXC |
| Sample type |
SRA |
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| Source name |
mouse embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell line: V6.5
cell type: mES
genotype/variation: Mll2-/-
chip antibody: Mll2 (Homemade, #9173)
rescue experiment: Halo-MLL2(ΔCXXC)
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| Treatment protocol |
Mouse ES cells were infected with lentivial shRNA against either Mll2 or Menin in the presense of 4ug/ml polybrene for 12 hours and then seleted with 1.5 ug/ml puromycinfor 48h before harvest for ChIP-seq or RNA-seq experiments. For rescue experiment, mouse ES cells were electroporated with indicated plasmids and selected with 400 µg/ml G418 for 3 days.
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| Growth protocol |
Mouse V6.5 ES cells were grown in DMEM supplemented with 15% FBS (HyClone), 2 mM L-glutamine, 0.1 mM nonessential amino acids (Stemcell Technologies) and 1,000 U/ml recombinant LIF (Millipore). Hanging drop method was used to form embryoid bodies (EBs) by culturing 1000 mouse ES cells in 25 µl differentiation medium (without LIF) on the lid of 150 mm culture dish for indicated days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ChIP assays were performed as previously described (Lee et al., 2006) . Briefly, 2-5x10^7 cells were crosslinked with 1% paraformaldehyde (Electron microscropy science #15710) at room temperature for 10 min and sonicated to generate chromatin fragments of 200-600bp. The sheared chromatin was immunoprecipated with a specific antibody. Total RNA was extracted with Trizol (Invitrogen #15596), treated with DNAase I (NEB #M0303) for 20 min at room temperature and purified with RNeasy Mini kit (Qiagen #74106).Circular Chromosome Conformation Capture (4C) technique was performed as described (van de Werken et al., 2012, Methods Enzymol). Cross-linked chromatin was digested with HindIII, ligated under diluted conditions. Cross links were reversed and DNA was further digested by NlaIII and again ligated under diluted conditions.For each viewpoint approximately 3.2 μg of the 4C-seq library was amplified using 16 individual PCR reactions with inverse primers containing Illumina adaptor sequence. Multiplexed samples were sequenced on the Illumina Nextseq500 system using 75 bp single-end reads according to the manufacturer’s specifications. 4C-seq reads were sorted, aligned, and translated to restriction fragments using the 4C-seq pipeline (van de Werken et al., 2012, Methods Enzymol).
ChIP-seq and RNA-seq libraries were constructed by following instructions in KAPA HTP Library Preparation Kits (Kapa Biosystems, #KK8234) and TruSeq Stranded Total RNA Library Prep kits with Ribo-Zero Human/Mouse/Rat (Illumina, #RS-122-2201 and #RS-122-2202), respectively. Indexed primers for ChIP-seq libraries are from Bioo Scientific (#514104 ). For 4C-seq, Cross-linked chromatin was digested with HindIII, ligated under diluted conditions. Cross links were reversed and DNA was further digested by NlaIII and again ligated under diluted conditions. For each viewpoint, approximately 3.2 μg of the 4C-seq library was amplified using 16 individual PCR reactions with inverse primers containing Illumina adaptor sequence. Multiplexed samples were sequenced on the Illumina Nextseq500 system using 75 bp single-end reads according to the manufacturer’s specifications.
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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 |
Sequencing data was acquired through the default Illumina pipeline using Casava v1.8.
ChIP-seq reads were aligned to the mouse genome (UCSC mm9) using the Bowtie aligner v0.12.9 allowing uniquely mapping reads only and allowing up to two mismatches (Langmead, 2009). RNA-seq reads were aligned to the mouse genome UCSC mm9 and to gene annotations from Ensembl 67 using TopHat v2.0.13 (Trapnell et al., 2009), using option –g 1 –x 1.
ChIP-seq reads were extended to 150 bases toward the interior of the sequenced fragment and normalized to total reads aligned (reads per million; RPM). RNA-seq reads were normalized to total reads aligned (reads per million; RPM) to generate bigWig files.
Peak detection was done using MACS v1.4.2 (Zhang et al., 2008). Associated control samples were used to determine statistical enrichment at p<1e-5. For down stream analysis, peaks were further filtered with p < 1e-8 and FDR < 0.05.
R package edgeR 3.0.8 was used to perform differential expression analysis at p-value < 1e-3 (Robinson et al., 2010).
Reads were demultiplexed by allowing no mismatch in library index and 1 mismatch in viewpoint sequences. The library index were trimmed before mapping. 4cseqpipe were used for alignment (van de Werken HG et al., 2012). The 4cseqpipe (van de Werken HG et al., 2012) generated figures directly from the alignment files.
Genome_build: mm9
Supplementary_files_format_and_content: BigWig
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| Submission date |
Feb 26, 2016 |
| Last update date |
May 15, 2019 |
| 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 |
GPL17021 |
| Series (1) |
| GSE78708 |
Not All H3K4 methylations are Created Equal: Mll2/COMPASS Dependency in Primordial Germ Cell Specification |
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| Relations |
| BioSample |
SAMN04517861 |
| SRA |
SRX1602650 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2073027_Mll2_Mll2KO_MLL2_deltaCXXC.bw |
179.9 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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