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Sample GSM5799599 Query DataSets for GSM5799599
Status Public on Feb 01, 2023
Title LZ-WT-noAb (non Antibody)
Sample type SRA
 
Source name no antibody
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 SAMN24811543
SRA SRX13705605

Supplementary file Size Download File type/resource
GSM5799599_5w-LZ-WT-noAb.macs2.bw 22.4 Mb (ftp)(http) BW
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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