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Sample GSM3902632 Query DataSets for GSM3902632
Status Public on Feb 12, 2020
Title H3K36me3.A1.rep2
Sample type SRA
Source name H3K36me3.A1
Organism Mus musculus
Characteristics strain: C57BL/6J (B6)
genotype/variation: wild type
cell type: homogenous synchronous spermatogenic cells
stage during meiotic process: A1
chip antibody: H3K36me3 (CST 4909S, lot 2)
Treatment protocol Briefly, 2-dpp mice were pipette fed 100 μg/g body weight WIN 18,446 (MP), suspended in 1% gum tragacanth, for 7 consecutive days. At Day 8 of WIN 18,446 treatments, these animals were received an i.p. injection of RA (Sigma; 30 μg/g body weight) in dimethyl sulfoxide (DMSO), and were then left to recover for sample collections. The undifferentiated spermatogonia was isolated from Lin28-YFP knockin mice. Testes from 3-week-old Lin28-YFP knockin mice were collected and digested by type I collagenase and 0.25% Trypsin as described previously1. After centrifugation, the pellet was resuspended in DMEM containing 2% BSA at a concentration of 1×106 cells/40 μL, followed by incubating with PE-conjugated anti-CD117 antibody (0.1 μg/106 cells; Molecular probes) for 30 min on ice. The undifferentiated spermatogonia (YFP-positive, PE-negative population) was collected using FACS (BD). The type A1 spermatogonia was isolated from synchronous Stra8-GFPCre mice. Testes were collected and digested. The type A1 spermatogonia (GFP-positive population) was collected using FACS. The type B spermatogonia and different stages of spermatocytes were isolated from synchronous Lin28-YFP and Vasa-tdTomato double positive mice. To isolate the type B spermatogonia, testes were digested and the synchronous advanced spermatogenic cells (tdTomato positive population) were collected using FACS. To isolate different stages of spermatocytes, testes were digested and the cell suspensions were stained with Hoechst 33342. The tdTomato positive 4N population were collected using FACS. The identity of sorted spermatocytes were validated using surface spreading and immunofluorescence.
Growth protocol Spermatogenesis was synchronized and validated as previously described.
Extracted molecule genomic DNA
Extraction protocol ChIP assay was carried out as previously described. The sorted homogenous synchronous spermatogenic cells were crosslinked with 1% formaldehyde for 10 mins and then stopped by adding 125 mM Glycine. Chromatin samples were lysed with lysis buffer (20 mM Tris-HCl pH8.0, 500 mM NaCl, 1 mM EDTA, 1% TritonX-100 and 0.1% SDS) and sonicated with Qsonica. Histone modification specific antibody was incubated with chromatin samples overnight at 4 ℃.0.5 ug spike-in antibody (Active motif #61686) and 25 ng spike-in chromatin (Active motif #53083) were used in this ChIP assay according to the manufacture’s guidelines.
DNA samples were analyzed using real time PCR and prepared for deep sequencing according to the manufacture’s guidelines (KAPA Biosystems KK8503 and VAHTS Universal DNA Library Prep Kit for Illumina V3 ND607). Finally, libraries were pooled and sequenced on the Illumina HiSeq 2500 sequencer for 150-bp paired-end sequencing.
Library strategy ChIP-Seq
Library source genomic
Library selection ChIP
Instrument model Illumina NovaSeq 6000
Data processing Base-calling was performed using CASAVA.
Firstly, All of the Histone modification ChIP-seq data were send to trim-galore to remove adapter contamination and low quality sequences, Phred score of any bases in a reads under 20 and adapter sequence were removed, reads after trimming shorter than 30 were discarded.
Trimmed sequences were then mapped with Bowtie (v2.2.6) using default parameters. Duplicate reads were removed by samtools rmdup -s. Unique and monoclonal mapped reads were extended to 150-bp based on the average sonicated chromatin DNA length.
Spike-in reads were mapped to drosophila genome (dm6) using same parameters.
GenomeCoverageBed (bedtools) was used to transfer bed files to bedGraph files, then spikein reads was used as a normalization factor that equalizes the signal across samples. Normalized bedGraph files were subsequently transferred to bigwig files.
Genome_build: mm10
Supplementary_files_format_and_content: bigWig&bed
Submission date Jun 24, 2019
Last update date Feb 13, 2020
Contact name Yuxuan Zheng
Organization name Fudan University
Street address 825 Zhangheng Rd.
City Shanghai
ZIP/Postal code 201203
Country China
Platform ID GPL24247
Series (1)
GSE132446 Refined spatial temporal epigenomic profiling reveals intrinsic connection between PRDM9-mediated H3K4me3 and the fate of double-stranded breaks
BioSample SAMN12127592
SRA SRX6355883

Supplementary file Size Download File type/resource
GSM3902632_H3K36me3.A1.rep2.bigWig 221.6 Mb (ftp)(http) BIGWIG
SRA Run SelectorHelp
Raw data are available in SRA
Processed data provided as supplementary file

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