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| Status |
Public on Aug 14, 2023 |
| Title |
FACS-sorted cKIT(+) spermatogonia, Cbx2[23KRA/23KRA], H3K27me3, rep1 |
| Sample type |
SRA |
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| Source name |
Testes
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| Organism |
Mus musculus |
| Characteristics |
tissue: Testes
cell type: cKIT(+) differentiating spermatogonia
strain: C57BL/6J
genotype: Cbx2[23KRA/23KRA]
cut&run antibody: H3K27me3 (CST, clone C36B11, Cat# 9733)
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| Treatment protocol |
No specific treatments
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| Growth protocol |
Mice were maintained in shoebox cages with standard chow diet.
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
FACS purified, >100,000 c-Kit(+) spermatogonia were attached to Poly-D-Lysine (R&D Systems, Cat# 3439-200-01) coated 24-well plates by 1 minute centrifugation at 80 × g (instead of binding to Concanavalin A beads). After the attachment, cells were fixed with 1% formaldehyde in PBS for 15 minutes. After two PBS washes and one PBST (0.05% Triton X-100 (v/v)) wash, cells were incubated with primary antibodies in PBST BSA (0.05% Triton X-100, 3% BSA) overnight at 4 ºC. Next day, cells were washed twice with PBST and finally washed with PBST EDTA (2 mM). Cells were then incubated with 500 ng/mL pA-MNase (protein A-MNase) in PBST BSA EDTA for more than 1 hour at 4 ºC. After pA-MNase binding, cells were washed with PBST EDTA twice, and the plate was placed on wet ice. Cells were then incubated in pre-chilled 200 µL PBST with 2 mM CaCl2 for 30 minutes. After MNase cutting, 200 µL stop solution (340 mM NaCl, 20 mM EDTA, 4 mM EGTA, 50 µg/µL RNase A) was added, and the plate was placed in a 37 ºC oven for 15 minutes. Released cut fragments were de-crosslinked in 0.1% SDS and 125 µg/mL Proteinase K (Sigma-Aldrich, Cat# 03115828001) in a 65 ºC oven for >12 hours. DNA was extracted using phenol-chloroform phase separation and sodium acetate/ethanol precipitation. Resuspended DNA was further cleaned up by using 2.0X Solid Phase Reversible Immobilization (SPRI) beads to remove residual salts.
Illumina sequencing libraries were generated based on a published protocol (Bowman et al., 2013) with the following modifications. SPRI beads were used in 2.0X ratio following end-repair and A-tailings steps instead of 1.8X. Adapters used in 2 nM final concentration instead of 10 nM. PCR extension was done for 30 seconds instead of 45 seconds. Final PCR cycles were between 12 to 17. Number of PCR cycles were determined to be in the exponential phase by a pre-run of 10% of reaction with SYBR green dye in a real time PCR machine.
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| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
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| Description |
CUT&RUN library
CBX2KRA_H3K27me3_rep1
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| Data processing |
Paired-end fastq reads were quality trimmed (score 20), with stringency 1 using trim_galore (ver. 0.4.3). Trimmed reads were mapped to the mm10 genome using bowtie2 (ver. 2.3.1) with the end-to-end option.
Mapped reads were qualify-filtered with MAPQ higher than 10, and only properly-paired reads were retained using Samtools (ver. 1.4.1). Picard (ver. 2.17.1) was used to remove duplicate reads (http://broadinstitute.github.io/picard).
Alignment files (bam) were converted to bigwig files using Deeptools (ver. 2.2.4) by normalizing with RPKM and with binsize 10.
Peaks were identified by using MACS2 (ver. 2. 1) with the ‘--broad' option, by comparing CUT&RUN results using CBX2 antibodies (Experimental) to IgG (Control) or using HA antibodies applied to Cbx2HA/HA (Experimental) or Cbx2+/+ (Control) mice.
All analysis scripts are available at (github.com/jongminkmg/cbx2).
Assembly: mm10
Supplementary files format and content: bigwig
Library strategy: CUT&RUN-seq
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| Submission date |
Aug 02, 2022 |
| Last update date |
Aug 14, 2023 |
| Contact name |
Jongmin J Kim |
| E-mail(s) |
[email protected]
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| Organization name |
Cornell University
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| Department |
Biomedical Sciences
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| Lab |
Kim lab
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| Street address |
930 Campus Road
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| City |
Ithaca |
| State/province |
NY |
| ZIP/Postal code |
14853 |
| Country |
USA |
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| Platform ID |
GPL21273 |
| Series (2) |
| GSE210367 |
Cell type-specific role of CBX2-cPRC1 at the onset of spermatogonial differentiation [CUT&RUN] |
| GSE210369 |
Cell type-specific role of CBX2-cPRC1 at the onset of spermatogonial differentiation |
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| Relations |
| BioSample |
SAMN30108989 |
| SRA |
SRX16775365 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6429286_CBX2KRA_H3K27me3_rep1.bw |
67.8 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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