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| Status |
Public on Dec 08, 2019 |
| Title |
TxCL63_UT_rep2_CutRun_Spen |
| Sample type |
SRA |
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| Source name |
ESC
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| Organism |
Mus musculus |
| Characteristics |
cell type: ESC
treatment: untreated
strain: BL/6J x CAST-EiJ
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| Treatment protocol |
24 hours later, cells were exposed to doxycycline (1ug/mL) and/or auxin (500uM). Cells were then collected at regular intervals.
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| Growth protocol |
Spen-degron mouse ES cells (TX1072) were seeded in 0.1% gelatin-coated T75 flasks (2.5*106 cells/flask). Cells were grown in 2i + LIF serum-containing ES cell medium - DMEM (Sigma), 15% FBS (Gibco), 0.1mM β-mercaptoethanol, 1000 U/ml leukemia inhibitory factor (LIF, Chemicon), CHIR99021 (3uM), PD0325901 (1uM) in 8% CO2 37°C incubators.
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| Extracted molecule |
total RNA |
| Extraction protocol |
10 million cells in suspension were fixed with 2% formaldehyde diluted in PBS for 10 minutes at room temperature. Fixation was quenched with 125mM glycine for 5 minutes and cells were washed twice with PBS. Fixed cells were then permeabilized with permeabilization buffer (20mM HEPES, 150mM NaCl, 0.5mM Spermidine, 0.25% TritonX-100, c0mplete EDTA free) for 5 minutes and washed twice in PBS. Cells were then resuspended in washing buffer (20mM HEPES, 150mM NaCl, 0.5mM Spermidine, 0.1% BSA, cOmplete EDTA free) and bound to concanavalin beads (50uL bead slurry used per 10 million cells), blocked in blocking buffer (wash buffer + 2mM EDTA) and incubated overnight with target antibodies diluted 1/200 in washing buffer on an end to end rotator. Cells were washed three times in washing buffer followed by 1-hour incubation with pA-MNase (1:400 in washing buffer) and washed again three times in washing buffer. After the last wash, cells were resuspended in washing buffer and left equilibrated to 0°C in a metal block for 10 minutes. To start digestion, CaCl2 was added to 1.5mM final concentration, taking care to return each sample to 0°C immediately after. Digestion was performed at 0°C for 1 hour, before being stopped by adding 2X-STOP solution (200mM NaCl, 20mM EDTA, 5mM EGTA, 0.1% NP-40, 40ug/mL glycogen). RNAse A was added to a final concentration of 50ug/mL and samples were incubated at 37°C for 20 minutes. SDS and proteinase K were then added to final concentrations of 0.1% and 300ug/mL respectively and samples were incubated at 56°C for 2 hours followed with 68°C for 16 hours to reverse crosslinking. Total DNA was extracted using phenol-chloroform followed by several rounds of ethanol precipitation and DNA size selection (using Ampure XP beads) to remove predominating large undigested DNA fragments. The selected small fragments (resulting from MNase digestion) were quantified and analyzed using Qubit and Tapestation assays.
Cut&Run libraries were prepared from 50ng DNA per samples, using the Accel-NGS 2S Plus DNA Library Kit (Swift) according to manufacturer’s protocol. Paired-end 100nt sequencing was performed on HiSeq2500 (Illumina, San Diego, CA).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Library strategy: CUT&RUN
All data were mapped to the mouse genome mm10, using the BL6-EiJ / CAST SNPs from the mouse genome project (v5 SNP142), and the gene annotation from ensembl (v92). Analyses were performed in R (v 3.4.2) and Bioconductor (v3.6).
Reads were trimmed using Trimgalore (v 0.4.4), mapped using STAR 10 (2.5.3a, parameters: --outFilterMultimapNmax 1 --outFilterMismatchNmax 999 --outFilterMismatchNoverLmax 0.06 --alignIntronMax 1 --alignMatesGapMax 2000 --alignEndsType EndToEnd --outSAMattributes NH HI NM MD), and removed when mapping to the mitochondrial genome. Remaining reads were split by allele using SNPsplit (v 0.3.2). Allele specific and the unassigned bam files were sorted, duplicates removed using picard (v2.18.2, parameters: REMOVE_DUPLICATES=true ASSUME_SORTED=true) and pooled as the total reads. BigWig of coverage files were done using DeepTools 11 bamCoverage (parameters: --extendReads --binSize 1, with --extendReads 200 for single end data). A scaling factor was calculated as 106 / total number of reads, and the same factor was given as the parameter --scaleFactor for both allelic signals.
Genome_build: mm10
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| Submission date |
May 27, 2019 |
| Last update date |
Dec 09, 2019 |
| Contact name |
samuel Collombet |
| E-mail(s) |
[email protected]
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| Organization name |
Ecole Normale Superieure
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| Department |
Biology
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| Lab |
Thieffry
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| Street address |
46 rue d'Ulm
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| City |
Paris |
| ZIP/Postal code |
75005 |
| Country |
France |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE131782 |
Spen regulates X chromosome inactivation (CUT&RUN) |
| GSE131784 |
Spen regulates X chromosome inactivation |
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| Relations |
| BioSample |
SAMN11866928 |
| SRA |
SRX5903675 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3819689_TxCL63_UT_rep2_CutRun_Spen.Aligned.out.merged.rmdup.bam.bw |
586.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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