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| Status |
Public on Jun 07, 2018 |
| Title |
ChIP-seq Smchd1-/- clone 1 H3K27me3 rep1 |
| Sample type |
SRA |
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| Source name |
neural progenitor cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: ESC-derived clonal neural progenitor cells
genotype: Smchd1-/-
antibodies: anti-H3K27me3 (Millipore 07-449, lot:2382150)
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| Growth protocol |
ES cell-derived neural progenitor cell clones were cultured on poly-L-ornithine (0.001%) and laminin (1µg/ml) coated plates with N2 media (DMEM/F-12, GlutaMAX supplement, 1X N2 supplement, 1X B27 supplement, 1X Pen/Strep) supplemented with FGF2 (20ng/ml), EGF (20ng/ml), laminin (1µg/ml), and heparin (10µg/ml). Undifferentiated ES cells were grown on 0.2% gelatin-coated plates with N2B27 media (mixture of 500ml DMEM/F12 media, 500ml Neurobasal media, 5ml N2 supplement, 10ml B27 supplement, 10ml Pen/Strep, 5ml GlutaMAX, and 2ml 55mM β-mercaptoethanol) supplemented with 1µM PD0325901, 3µM CHIR-99021, and 1000U/ml LIF. Embryoid bodies (EBs) were grown with differentiation media (DMEM, high glucose, GlutaMAX supplement, pyruvate, 15% Hyclone FBS, 25mM HEPES pH 7.2-7.5, 1X MEM non-essential amino acids, 1X Pen/Strep, 0.1mM β-mercaptoethanol) in suspension (D4 EBs) or on gelatin-coated plates (D7 EBs).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total RNA was extracted using standard Trizol protocol. ChIP/CHART-enriched and input DNA were extracted with phenol:chloroform.
RNA-seq libraries were generated by dUTP-mediated directional RNA-seq based on NEBNext Ultra directional RNA library preparation protocol. ChIP-seq and Hi-C libraries were generated by NEBNext ChIP-Seq Library Prep Master Mix Set for Illumina. CHART-seq libraries were generated by NEBNext Ultra DNA Library Prep Kit for Illumina.
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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 |
ChIP-seq: Input and ChIP-seq reads were aligned allele-specifically to 129S1/SvJm (mus) and CAST/Eih (cas) genome using NovoAlign_v3-1.00.02. After removing PCR duplicates, uniquely mapped reads were used to generate fpm-normalized bigWig files from all reads (comp), mus-specific (mus) reads, and cas-specific (cas) reads. H3K27me3 ChIP-seq tracks were further scaled by total reads mapped to the X chromosome.
Genome_build: mm9
Supplementary_files_format_and_content: ChIP-seq: Uniquely mapped reads without PCR duplicates were used to generate fragment-per-million (fpm)-normalized ChIP-seq coverage files (bigWig). WT1, WT clone 1. KO1, Smchd1-/- clone 1. rep1, replicate 1. rep2, replicate 2. cas, cas-specific reads. mus, mus-specific reads. comp, all reads. To compare the distribution of H3K27me3 marks between WT and Smchd1-/-, we scaled them based on the number of total uniquely mapped reads on the X chromosome (WT1.ChIP.H3K27me3.rep1.comp.scaled.bw and KO1.ChIP.H3K27me3.rep1.comp.scaled.bw). Using chromatin signatures and expression profiles, we defined 6 classes of X-linked genes (X-linked.gene.classes.bed): silent genes (black), escapees (pink), Class I genes (red), Class II genes (orange), Class III genes (blue), and unclassified genes (gray). We also compiled a list of escapees reported in the literature (allreportedescapees.bed and all_reported_escapees.xlsx) to investigate the relationship between Class I genes and escapees. For CTCF and RAD21, we called peaks using MACS2, and merged the peaks called in WT and Smchd1-/- for analysis (CTCF.peaks.rep1.bed and RAD21.peaks.rep1.bed).
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| Submission date |
Jun 13, 2017 |
| Last update date |
Jun 07, 2018 |
| Contact name |
Chen-Yu Wang |
| E-mail(s) |
cywang@molbio.mgh.harvard.edu
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| Organization name |
Massachusetts General Hospital
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| Department |
Molecular Biology
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| Lab |
Jeannie T. Lee
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| Street address |
185 Cambridge Street
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| City |
Boston |
| State/province |
MA |
| ZIP/Postal code |
02114 |
| Country |
USA |
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| Platform ID |
GPL17021 |
| Series (1) |
| GSE99991 |
Investigating the role of SMCHD1 in de novo X chromosome inactivation |
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| Relations |
| BioSample |
SAMN07230066 |
| SRA |
SRX2916327 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2667237_KO1.ChIP.H3K27me3.rep1.cas.bw |
132.4 Mb |
(ftp)(http) |
BW |
| GSM2667237_KO1.ChIP.H3K27me3.rep1.comp.bw |
1.0 Gb |
(ftp)(http) |
BW |
| GSM2667237_KO1.ChIP.H3K27me3.rep1.comp.scaled.bw |
1.0 Gb |
(ftp)(http) |
BW |
| GSM2667237_KO1.ChIP.H3K27me3.rep1.mus.bw |
549.1 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
| Processed data are available on Series record |
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