|
| Status |
Public on May 09, 2023 |
| Title |
WT-MNASE-3 |
| Sample type |
SRA |
| |
|
| Source name |
epididymis and vas deferens
|
| Organism |
Mus musculus |
| Characteristics |
tissue: epididymis and vas deferens
cell type: sperm
genotype: wild type
treatment: MNASE digestion
|
| Treatment protocol |
30 million sperm per genotype were treated with 5 µl of 20 mg/ml DNaseI for 15 minutes at 37°C. Sperm pellets were then washed several times in PBS and subsequently treated with 0.5% Triton and 0.05% L-alpha-phosphatidylcholine for 15 minutes on ice.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Sperm pellets were then centrifuged at 2500xg for 5 minutes and chromatin was then decondensed using 50 mM DTT for 1 hour at room temperature. DTT was then quenched for 30 minutes at room temperature with 100 mM N-ethylmaleimide. Sperm cells were then lysed in buffer containing 0.25% NP-40 and 0.25% DOC for 30 minutes at room temperature prior to addition of 5 U MNase. Samples were digested at 37oC for 5 minutes, centrifuged at 20,000xg for 20 minutes and soluble chromatin was collected. Insoluble chromatin was digested a second time with 15 U of MNase at 37oC for 5 minutes. Samples were then run on a 2% agarose gel and bands corresponding to Mononucleosomes were cut out and purified using a Zymo gel extraction kit for sequencing.
Libraries constructed with the NEBNext Ultra II DNA Library Prep Kit are constructed with UMI adaptors. The unique molecular identifier consists of an 11 base sequence that is located 3’ to the i7 index. This sequence enables more accurate removal of duplicate reads and error correction through consensus sequence building. Sequence data containing a UMI read will be recorded as Read 2 in the FASTQ files, with the forward and reverse data being reported as Read 1 and Read 3, respectively. For all paired-end analysis, R1 was used as Forward read and R3 was used as Reverse read.
Mnase-seq using NovaSeq S4 Illumina Sequencing platform
|
| |
|
| Library strategy |
MNase-Seq |
| Library source |
genomic |
| Library selection |
MNase |
| Instrument model |
Illumina NovaSeq 6000 |
| |
|
| Description |
wild type
|
| Data processing |
CTA and fastQC
BWA-MEM for alignment
Picard MarkDuplicates and Samtools for filtering
MACS2 callpeak
Assembly: mm10
Supplementary files format and content: Bigwig trakc files and tab-delimited broadPeak files
|
| |
|
| Submission date |
Feb 14, 2023 |
| Last update date |
May 09, 2023 |
| Contact name |
Qianyi Ma |
| E-mail(s) |
qzm100@gmail.com
|
| Organization name |
University of Michigan
|
| Department |
Human Genetics
|
| Street address |
1141 E Catherine St
|
| City |
Ann Arbor |
| State/province |
MI |
| ZIP/Postal code |
48109 |
| Country |
USA |
| |
|
| Platform ID |
GPL24247 |
| Series (2) |
| GSE225268 |
Sperm chromatin structure and reproductive fitness are altered by substitution of a single amino acid in mouse Protamine 1 [MNase-Seq] |
| GSE225271 |
Sperm chromatin structure and reproductive fitness are altered by substitution of a single amino acid in mouse Protamine 1 |
|
| Relations |
| BioSample |
SAMN33285330 |
| SRA |
SRX19364166 |
