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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 13, 2021 |
| Title |
EPCAM_rep2_IgG |
| Sample type |
SRA |
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| Source name |
EPCAM+ endometrial epithelial cells
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| Organism |
Mus musculus |
| Characteristics |
strain: CD-1
genotype: Wild-type
antibody: Rabbit IgG (2729, Cell Signaling)
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| Treatment protocol |
Mouse uteri were surgically removed and minced using scissors. Tissues were digested using the MACS Multi Tissue Dissociation Kit II (Miltenyi Biotec). Endometrial epithelial cells were magnetically sorted using EpCAM-PE and anti-PE microbeads.
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| Growth protocol |
Mice were housed at the Van Andel Research Institute Animal Facility and the Michigan State University Grand Rapids Research Center in accordance with protocols approved by the Van Andel Research Institute and Michigan State University, under AUF# 01/16-099-00.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
Concanavalin A magnetic beads (Bangs) were washed in Binding Buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 1 mM CaCl2, 1 mM MnCl2) and incubated with either anti-ARID1A (D2A8U, Cell Signaling) or Rabbit IgG (2729, Cell Signaling). Purified cells were washed in Wash Buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 5 mM sodium butyrate) then added to the conjugated antibody-bead slurry. After 10 minutes of nutating incubation at ambient temperature, Antibody Buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 5 mM sodium butyrate, 2 mM EDTA, 0.05% digitonin) was added to cell bead mixtures, and nuclear permeabilization was confirmed with Trypan blue dye. Reactions then incubated overnight at 4°C. Reactions were washed with Digitonin Buffer (20 mM HEPES-NaOH pH 7.5, 150 mM NaCl, 0.5 mM spermidine, 5 mM sodium butyrate, 0.05% digitonin) and incubated with pAG-MNase (CUTANA, EpiCypher) for one hour at room temperature, followed by an additional wash in Digitonin Buffer then Low-Salt Rinse Buffer (20 mM HEPES-NaOH pH 7.5, 0.5 mM spermidine, 5 mM sodium butyrate). Calcium Incubation Buffer (3.5 mM HEPES-NaOH pH 7.5, 10 mM CaCl2, 0.05% digitonin) was then added to activate the pAG-MNase enzyme, and the reaction was quenched after 3 minutes using EGTA-STOP buffer (170 mM NaCl, 20 mM EGTA, 0.05% digitonin, 20 µg/mL RNase A, 20 µg/mL glycogen, 0.8 pg/mL Saccharomyces cerevisiae nucleosomal DNA as spike-in). Fragments were then released into solution at 37°C followed by 5 minutes centrifugation at 16,000 x g. Eluted DNA were then purified with the NucleoSpin Gel and PCR Clean-up Kit (Takara).
Libraries for CUT&RUN samples were prepared by the Van Andel Genomics Core from 0.5-1 ng of IP material, using the KAPA Hyper Prep Kit (v5.16) (Kapa Biosystems). Prior to PCR amplification, end-repaired and A-tailed DNA fragments were ligated to Bioo Scientific NEXTflex Adapters (Bioo Scientific) at a concentration of 500 nM. Quality and quantity of the finished libraries were assessed using a combination of Agilent DNA High Sensitivity chip (Agilent Technologies, Inc.), QuantiFluor dsDNA System (Promega Corp.), and Kapa Illumina Library Quantification qPCR assays (Kapa Biosystems). Individually indexed libraries were pooled and 50 bp, paired end sequencing was performed on an Illumina NovaSeq6000 sequencer using an S1, 100 cycle sequencing kit (Illumina Inc.) Each library was sequenced to an average depth of 50 million reads. Base calling was done by Illumina RTA3 and output was demultiplexed and converted to FastQ format with Illumina Bcl2fastq v1.9.0.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Description |
IgG CUT&RUN library from 100,000 sorted EPCAM+ endometrial epithelial cells (biological replicate #1) from adult, wild-type mice.
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| Data processing |
library strategy: CUT&RUN
Raw paired-end reads for anti-ARID1A or IgG CUT&RUN were trimmed with cutadapt and Trim Galore! and analyzed for quality via FastQC.
Trimmed reads were aligned to mm10 genome assembly with bowtie2 using flag `--very-sensitive` and filtered for only properly-paired reads with samtools using flag `-f 3`.
Picard MarkDuplicates (http://broadinstitute.github.io/picard/) was used to remove PCR duplicates.
For each biological replicate, MACS2 was used to call ARID1A broad peaks against the respective IgG negative control as input, with FDR < 0.25 threshold.
The resulting peaks were repeat-masked by ENCODE blacklist filtering and filtered for non-standard contigs.
A naive replicate-overlapping peak set was constructed by calling peaks on pooled replicates followed by bedtools intersect to select for peaks of at least 50% overlap with each biological replicate.
Genome_build: mm10
Supplementary_files_format_and_content: Tabular peak set files including blacklist-filtered CUTNRUN replicate broadPeaks (FDR < 0.25 MACS2 output) and naive replicate-overlapping broadPeaks.
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| Submission date |
Sep 20, 2021 |
| Last update date |
Dec 13, 2021 |
| Contact name |
Ronald L. Chandler |
| E-mail(s) |
rlc@msu.edu
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| Organization name |
Michigan State University
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| Department |
Obstetrics, Gynecology and Reproductive Biology
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| Street address |
3100-4 400 Monroe NW
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| City |
Grand Rapids |
| State/province |
MI |
| ZIP/Postal code |
49503 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE184498 |
Co-existing TP53 and ARID1A mutations promote aggressive endometrial tumorigenesis [CUT&RUN] |
| GSE184499 |
Co-existing TP53 and ARID1A mutations promote aggressive endometrial tumorigenesis |
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| Relations |
| BioSample |
SAMN21528991 |
| SRA |
SRX12277397 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data not provided for this record |
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