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GEO help: Mouse over screen elements for information. |
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| Status |
Public on May 03, 2019 |
| Title |
Hi-C D4 EB [re-analyzed GSM3036557] |
| Sample type |
SRA |
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| Source name |
mouse embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryonic stem cells
genotype: WT
gender: female
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| Treatment protocol |
To treate cells with 5-aza-2'-deoxycytidine (Aza, A3656, Sigma), MEFs were grown in MEF media containing either DMSO (control) or Aza (0.3μM), with media changed daily for 5 days. To deplete PRC1, MEFs were transfected with either scramble siRNAs (siGENOME Non-Targeting siRNA #1, D-001210-01, Dharmacon) or a mixture of siRNAs targeting RING1A (D-062335-03, Dharmacon) and RING1B (D-042180-01, Dharmacon) using Lipofectamine® RNAiMAX Transfection Reagent (13778075, Thermo Fisher Scientific) according to manufacturer's instructions. Transfection of siRNAs was performed once a day for 4 days.
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| Growth protocol |
MEFs were grown in MEF media [DMEM, high glucose, GlutaMAX supplement, pyruvate (10569044, Thermo Fisher Scientific), 10% FBS, 25mM HEPES pH 7.2-7.5 (15630130, Thermo Fisher Scientific), 1X MEM non-essential amino acids (11140076, Thermo Fisher Scientific), 1X Pen/Strep (15140163, Thermo Fisher Scientific), and 0.1mM β-mercaptoethanol (21985023, Thermo Fisher Scientific)].
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Total RNA was extracted using standard Trizol protocol. ChIP/CHART/Hi-C-enriched and input DNA were extracted with phenol:chloroform.
RNA-seq libraries were prepared by dUTP-mediated directional RNA-seq following NEBNext Ultra directional RNA library preparation protocol. ChIP-seq and Hi-C libraries were prepared 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.
Paired-end 50 or 69-cycle with Illumina HiSeq
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 2500 |
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| Data processing |
Library strategy: Hi-C
RNA-seq: Reads were aligned allele-specifically to 129S1/SvJm (mus) and CAST/Eih (cas) genome using TopHat2.0.10. After removal of PCR duplicates, all unique reads mapped to the exons of each gene were quantified by Homer4.8 to generate a raw read count table. Strand-resolved fpm-normalized bigWig files from the raw RNA-seq reads for all reads (comp), mus-specific (mus) reads, and cas-specific (cas) reads were generated using custom scripts.
ChIP-seq: Reads were first trimmed to 50bp if performed using pair-end 69 cycles. 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. For the input sample of KO cells, libraries were sequenced twice to reach the desired depth, and the two sequencing files were combined to generate the fpm-normalized bigWig files. To quantify the difference in H3K27me3 and H2AK119ub enrichment between WT and KO cells, we subtracted the ChIP-seq profile of WT cells from that of KO cells using bigwigCompare.
CHART-seq: Input and CHART-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 input-subtracted coverage profiles using SPP. SPP-normalized profiles were then scaled between WT and KO cells so that they have equal total Xist coverage on the X chromosome. To quantify the difference in Xist enrichment between WT and KO cells, we subtracted the Xist CHART-seq profile of WT cells from that of KO cells using R.
In situ Hi-C: Each end of the read-pairs was aligned separately as single-end reads to 129S1/SvJm (mus) and CAST/Eih (cas) genome using NovoAlign_v3-1.00.02 and allele-specificity was assigned. The single-end alignments were joined to generate a Hi-C summary file. For the replicate 2 of KO cells treated with scramble siRNAs, libraries were sequenced twice to reach the desired depth, and the two sequencing files were combined to generate the Hi-C summary files.
Genome_build: mm9
Supplementary_files_format_and_content: RNA-seq: Uniquely mapped reads without PCR duplicates were used to generate strand-resolved fragment-per-million (fpm)-normalized RNA-seq coverage files (bigWig). WT1, WT clone 1. WT2, WT clone 2. KO1, Smchd1-/- clone 1. KO2, Smchd1-/- clone 2. DMSO, DMSO-treated. Aza, Aza-treated. cas, cas-specific reads. mus, mus-specific reads. comp, all reads. +, forward strand. -, reverse strand. Raw exonic reads in each RNA-seq library were quantified using Homer and summarized in RNAseq_raw_read_count_table.txt. In this table, purecas.TTF data were obtained by re-analyzing published pure cast tail-tip fibroblast RNA-seq data (GSM1412989) in Pinter et al., 2015. Purecas.MEF data were from pure cas MEF RNA-seq data in this GEO entry.
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). WT, WT clone 1. KO, Smchd1-/- clone 1. cas, cas-specific reads. mus, mus-specific reads. comp, all reads. delta: KO minus WT. rep1, replicate 1. rep2, replicate 2.
Supplementary_files_format_and_content: CHART-seq: Uniquely mapped reads without PCR duplicates were used to generate fragment-per-million (fpm)-normalized CHART-seq coverage files (bigWig). WT, WT clone 1. KO, Smchd1-/- clone 1. cas, cas-specific reads. mus, mus-specific reads. comp, all reads. Uniquely mapped comp reads without PCR duplicates were then used to generate the input-subtracted Xist coverage profile by SPP as described in Simon et al., 2013 and scaled between WT and KO cells (files whose name starts with “SPP.normalized”). delta: KO minus WT. rep1, replicate 1. rep2, replicate 2.
Supplementary_files_format_and_content: In situ Hi-C: Allele-resolved Hi-C data were presented as Hi-C summary file (file names ending with “.summary.txt”), where the first 7 columns represent read names, chromosome for read 1, positions for read 1 (5’ end of read), strand of read 1 (+ or -), chromosome for read 2, positions for read 2 (5’ end of read), and strand of read 2 (+ or -), respectively. WT, WT clone 1. KO, Smchd1-/- clone 1. Scramble, scramble siRNA-treated. PRC1_KD, RING1A and RING1B siRNA-treated. HNRNPK_KD, HNRNPK siRNA-treated. cas, cas-specific reads. mus, mus-specific reads. comp, all reads. rep1, replicate 1. rep2, replicate 2. To match sequencing depths between samples, Hi-C summary files were randomly sampled to generate down-sampled Hi-summary files (file names starting with “Downsampled”). Principle component analysis was performed at 200-kb resolution using Homer. We also similarly re-analyzed published Hi-C data (GSE99991, GSE67516) of undifferentiated female ES cells (D0.ES, GSM3036556), day 4 embryoid bodies (D4.EB, GSM3036557), and day 7 embryoid bodies (D7.EB, GSM3036558) to derive the PC1 values of the X chromosome at 200-kb resolution using Homer. Principle component 1 or 2 were presented as bedGraph files (file names ending with “.PC1.200kb.bedGraph” or “.PC2.200kb.bedGraph”). Insulation scores were computed using iteratively corrected Hi-C contact matrices at 100-kb resolution and summarized in X_chromosome.insulation_score.txt. To investigate the relationships between TAD boundaries and borders of chromosome compartments, we additionally performed PCA at 100-kb resolution using Homer on the Hi-C data of WT and Smchd1-/- MEFs. Xa, comp (D0.ES) or cas (all others). Xi, mus.
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| Submission date |
Apr 15, 2019 |
| Last update date |
May 05, 2019 |
| 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) |
| GSE116413 |
Unfolding the inactive X chromosome |
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| Relations |
| Reanalysis of |
GSM3036557 |
| BioSample |
SAMN11419959 |
| SRA |
SRX5688395 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3722628_D4.EB.Xa.PC1.200kb.bedGraph.gz |
9.9 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM3722628_D4.EB.Xi.PC1.200kb.bedGraph.gz |
9.9 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Processed data provided as supplementary file |
| Raw data are available in SRA |
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