 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Feb 01, 2023 |
| Title |
LZ-WT2 (S9.6 CUT&Tag) |
| Sample type |
SRA |
| |
|
| Source name |
S9.6 CUT&Tag
|
| Organism |
Mus musculus |
| Characteristics |
strain: C57BL/6
tissue: Testis
cell type: Leptotene/zygotene
genotype: Wild-type
|
| Treatment protocol |
Mice were kept under steady state, or subjected to different stress conditions such as aging, rosiglitazone diet feeding, sub-lethal irradiation, or mid-diaphyseal femur fracture.
|
| Growth protocol |
All mice were kept in an SPF facility operated in a 12-hour light/dark cycle.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
After removal of the tunica albuginea, the testes were incubated in 5mL of DPBS containing 120 U/mL of collagenase type I at 32 °C with gentle agitation for 8~10 min until seminiferous tubules were dispersed, The dispersed seminiferous tubules were further digested with 5 ml 0.25% trypsin, plus 0.1 ml 5mg/ml DNase I at 32°C for 8 min. The digestion was terminated by adding 0.5 mL FBS to inactivate trypsin. The cell suspension was filtered through a 70 μm cell strainer and then centrifuged at 500 g for 5 min at 4 °C. The cells’ pellet was resuspended at a concentration of 106 cells/ml in DMEM with Hoechst 33342 (5 μg/106 cells) and 5 μl DNase I, followed by cell suspension was rotated for 30min at 32 °C with gentle rotation. Centrifuged at 500 g for 5 min at 4 °C to remove supernatant. Immediately before sorting, the cells were stained with propidium iodide (1 μg/106 cells) at 25 °C and filtered with a 40 μm cell strainer.
5 × 10^4 spermatogonia were sorted and washed once in 0.5 ml wash buffer [20 mM Hepes (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, and 1× protease inhibitors], and were incubated with 10 μl pre-washed concanavalin A–coated magnetic beads in a 1.5 ml microtube at 25 °C for 10 min. Cells were resuspended in 80 μl ice-cold antibody incubation buffer [20 mM Hepes (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, 1× protease inhibitors, 0.025% digitonin, 0.01% NP-40, and 2 mM EDTA] with S9.6 antibody (MABE1095, Millipore) at 1:100 dilution, and then rotated end-over-end at 4 °C overnight. Subsequently, cells were resuspended in 100 μl dig-wash buffer [20 mM Hepes (pH 7.5), 150 mM NaCl, 0.5 mM spermidine, 1 × protease inhibitors, and 0.025% digitonin] with rabbit anti-mouse IgG (Ab6709, Thermo Fisher) at 1:100 dilution and rotated end-over-end at 25 °C for 1 h. After washing, home-made pA-Tn5 adapter complexes in dig-300 buffer [20 mM Hepes (pH 7.5), 300 mM NaCl, 0.5 mM spermidine, 1 × protease inhibitors, and 0.01% digitonin] were added to samples at a final concentration of 1:100 and then rotated end-over-end at 25 °C for 1 h. After washing, cells were resuspended in 300 μl tagmentation buffer [dig-300 buffer with 10 mM MgCl2] and incubates at 37 °C for 1 h. To stop tagmentation and solubilize DNA fragments, 10 μl 0.5M EDTA, 3 μl 10% SDS and 2.5 μl 20 mg/mL Proteinase K were added to each sample and incubated at 50 °C for 1 h. After extraction with phenol-chloroform and ethanol precipitation, libraries were amplified by PCR. The barcoded libraries were pooled and sequenced on the Illumina HiSeq X Ten platform to generate 150 bp paired-end reads.
|
| |
|
| Library strategy |
OTHER |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
HiSeq X Ten |
| |
|
| Data processing |
Library strategy: CUT&Tag
R-loop CUT&Tag raw data was trimmed to filter low-quality bases using TrimGalore.
Trimmed reads were aligned to mm9 genome reference (UCSC) using Bowtie2 aligner
For unique alignments, duplicate reads were marked by “MarkDuplicates” function of Picard software.
The coverage of R-loop CUT&Tag data was calculated and normalized by RPKM using deepTools.
Uniquely mapped reads were used to carry out peak calling using MACS2.
Genome_build: mm9
Supplementary_files_format_and_content: bigwig files to perform visualization
|
| |
|
| Submission date |
Jan 10, 2022 |
| Last update date |
Feb 02, 2023 |
| Contact name |
Heng-yu Fan |
| E-mail(s) |
Fanhengyu_lab@163.com, 21907079@zju.edu.cn
|
| Organization name |
Life Sciences Institute
|
| Lab |
Fan heng-yu
|
| Street address |
Yuhangtang Road
|
| City |
Hangzhou |
| State/province |
Zhejiang |
| ZIP/Postal code |
310058 |
| Country |
China |
| |
|
| Platform ID |
GPL21273 |
| Series (1) |
| GSE193401 |
Genome-wide Map of R-loops Reveals its interplays with Transcription and Genome Integrity during Germ Cell Meiosis |
|
| Relations |
| BioSample |
SAMN24811553 |
| SRA |
SRX13705609 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5799589_5w-LZ-WT2.macs2.bw |
32.4 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
