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| Status |
Public on Dec 13, 2023 |
| Title |
Mouse Male MEF – XIST Pulldown |
| Sample type |
SRA |
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| Source name |
Somatic
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| Organism |
Mus musculus |
| Characteristics |
cell type: Somatic
library strategy: RAP-Seq
experiment type: XIST RAP
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| Treatment protocol |
In addition to growing each cell type in its appropriate media condition as described under "growth protocol", cells were not treated with any additional agent(s).
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| Growth protocol |
Mouse embryonic stem cells (ESCs) were grown in mouse ESC medium containing knockout DMEM (Life Technologies), 15% FBS (Omega), 2mM L-glutamine (Life Technologies), 1x NEAA (Life Technologies), 0.1mM β-Mercaptoethanol (Sigma), 1x Penicillin/Streptomycin (Life Technologies) and 1000U/mL mouse LIF (homemade). To derive epiblast-like cells (EpiLCs), ESCs were first adjusted for at least 3 passages under feeder-free conditions on 0.5% gelatin-coated plates in the presence of 1000U/mL LIF and two inhibitors, CHIR99021 (3 μM) and PD0325901 (0.4 μM) (henceforth referred to as 2i+LIF) in serum-free N2B27 medium containing 1x N2 supplement and 1x B27 supplement (ThermoFisher), 2mM L-glutamine (Life Technologies), 1x NEAA (Life Technologies), 0.1mM β-Mercaptoethanol (Sigma) and 0.5 x Penicillin/Streptomycin (Life Technologies). Next, to induce EpiLC differentiation, cells were dissociated with accutase and seeded at a density of 20,000 cells/mL in N2B27 media supplemented with 20 ng/mL Activin A and 12 ng/mL bFGF on geltrex-coated plates. Fresh media was exchanged daily for both ESCs and EpiLCs. Mouse embryonic fibroblasts (MEFs) were cultured on non gelatin-coated plates under feeder-free conditions in DMEM (Life Technologies), 15% FBS (Omega), 2mM L-glutamine (Life Technologies), 1x NEAA (Life Technologies), 0.1mM β-Mercaptoethanol (Sigma) and 1x Penicillin/Streptomycin (Life Technologies). Fresh media was exchanged once every 3 days.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
5× 106cells were harvested per condition after accutase-mediated dissociation and RAP-seq was performed, based on the protocol adapted from Engreitz et al. (Engreitz et al., 2013). Briefly, cells were incubated for 45min with freshly made 2mM DSG in PBS at room temperature, crosslinked with 3% formaldehyde for 10min and quenched with 500mM glycine. Cells were subsequently pelleted at 1,500xg for 5min at 4˚C, aliquoted into vials of 10 million cells each, and subjected to lysate preparation as follows: Cell pellets were resuspended in 10ml of nuclear extraction buffer LB1 containing50mM HEPES-KOH (pH 7.5), 140mM NaCl, 1mM EDTA, 10% (vol/vol) glycerol, 0.5% (vol/vol) NP-40/Igepal CA-630 and 0.25% (vol/vol) Triton X-100, and incubated for 10min with rotation at 4°C, then pelleted at 2000xg for 5min and the same procedure was performed with buffer LB2, which contains 10mM Tris-HCL (pH 8.0), 200mM NaCl, 1mM EDTA and 0.5mM EGTA. For cell lysis, nuclei were resuspended in 300μL of buffer LB3 containing 10mM Tris-HCl (pH 8.0), 100mM NaCl, 1mM EDTA, 0.5mM EGTA, 0.1% (wt/vol) sodium deoxycholate and 0.5% (vol/vol) N-lauroylsarcosine, then sonicated on ice using the Misonix S-400 sonicator with microtip (model number: U1240A0418) for 2min at an amplitude of 12 with 1sec pulses intermitted by 3sec pauses. Next, chromatin was further digested using TURBO DNase at a concentration of 0.1-0.4U/μl at 37°C for 15min. The digestion was stopped by the addition of DNase Stop Solution (mixture of EDTA and EGTA). 5% of each sample was set aside as the input control, while the remaining sample was used for the actual pulldown. The RNA pulldown was performed using 50pmol of 90nt-long biotinylated oligonucleotide probes for every 5× 106cells and Streptavidin C1 beads. DNA was eluted by RNase H digestion, and the crosslinks were reversed by proteinase K digestion of the eluted DNA at 60°C.
DNA libraries were prepared using NEB Next Ultra End Repair/dA-Tailing Module (NEB) and TruSeq DNA adapters (Illumina) were ligated using Quick Ligase (NEB). Libraries were amplified by KAPA HiFi Polymerase, pooled and sequenced on the Illumina HiSeq 6000 platform to generate 50bp pair-end reads.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
RAP-Seq using probes targeting Xist in Male MEFs
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| Data processing |
Trim: trim_galore (v0.4.1) with default parameters was used to trim DNA sequencing reads
Align: Bowtie2 (v2.2.9) with the default parameters was used to align reads
Filter: Samtools (v1.9) was used to remove reads with mapping quality less than 30, or marked by Picard MarkDuplicates (v2.1.0) as PCR duplicates
Count: Bedtools (v2.26.0) intersect was used to count reads in each genomic region (either 100kb windows every 25kb or 1Mb every 250kb)
Enrichment: read counts in each window were normalized by the total read counts in each sample. The ratio of the normalized reads in the pulldown to the input were used as inputs
Peaks: MACS2 (v2.2.7.1) using broad and max-gap=100 was used to call peaks
Assembly: mm9
Supplementary files format and content: bedgraph files represent normalized enrichment ratios in windows across the genome (either 100kb windows every 25kb or 1Mb every 250kb). Is_unmappable=yes mark unmappable genomic regions
Supplementary files format and content: peak files represent broad peaks called for XIST pulldown files samples compared to input
Library strategy: RAP-seq
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| Submission date |
Aug 22, 2023 |
| Last update date |
Dec 13, 2023 |
| Contact name |
Kathrin Plath |
| Organization name |
UCLA
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| Street address |
BOX 951737, 36-133 CHS
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| City |
Los Angeles |
| State/province |
CA |
| ZIP/Postal code |
90095-1737 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE241440 |
XIST directly regulates X-linked and autosomal genes in naïve human pluripotent cells [RAP-seq_mouse] |
| GSE241444 |
XIST directly regulates X-linked and autosomal genes in naïve human pluripotent cells |
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| Relations |
| BioSample |
SAMN37116366 |
| SRA |
SRX21457659 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7729037_Autosomal_MEF_Male_1000.broadPeak.gz |
6.9 Kb |
(ftp)(http) |
BROADPEAK |
| GSM7729037_XIST_vs_Input_MEF_Male_pulldown_100kb_25kb.bedgraph.gz |
606.8 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM7729037_XIST_vs_Input_MEF_Male_pulldown_1Mb_250kb.bedgraph.gz |
51.4 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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