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| Status |
Public on May 28, 2020 |
| Title |
Day3_Dox-minus-1A-MYB-plus-2 |
| Sample type |
SRA |
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| Source name |
mouse J1 embryonic stem cells
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| Organism |
Mus musculus |
| Characteristics |
no. of passages and ages of weeks: P36
background strain: 129S4/SvJae
chip antibody: none
genotype: J1 ESC Tn(dCas9-VPR/RLTR10B2-gRNA-DsRed)
agent: Dox-, A-MYB
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| Treatment protocol |
In ES cell culture, doxycycline (1 µg/ml) and A-MYB expression were induced at day1 and day2
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| Growth protocol |
For RNA-seq analysis, ES cells were cultured in ESC media (15% FBS, 25 mM HEPES, 1× GlutaMAX, 1× MEM non-essential amino acids, 1× Penicillin/Streptomycin and 0.055 mM β-Mercaptoethanol in DMEM High Glucose (4.5g/l)) with 2i (PD0325901, 1 µM; LC Laboratories and CHIR99021, 3µM, LC Laboratories) and LIF (1300 U/ml, in-house) on cell culture plates coated with 0.2% gelatin, under feeder-free condition. For native ChIP-seq analysis in pachytene spermatocytes, from WT and A-myb mutant testes, we prepared testicular cell suspension from 1 male mouse at ages of 8-12 weeks, and isolated pachytene spermatocytes according to small-scale STA-PUT method described in our previous report (Adam SR, PLoS Genet, 2018).
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| Extracted molecule |
total RNA |
| Extraction protocol |
For RNA-seq analysis, total RNA from ES cells on each well of 24 well plate was extracted using an RNeasy Plus Mini Kit (QIAGEN), with gnomic DNA elimination. For ChIP-seq analysis, to extract chromatin, isolated pachytene spermatocytes were suspended in 20 µl of Nuclei EZ Lysis Buffer (SIGMA), and extensively digested chromatin with 2 IU/µl Micrococcal Nuclease (MNase, NEB) at 37℃ for 5 min.
RNA-seq library preparation was carried out using TruSeq Stranded mRNA Library Prep kit (Illumina) following manufacturer’s instructions. ChIP-seq library preparation was carried out by NEBNext Ultra II DNA Library Prep Kit (NEB) following manufacturer’s instructions.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Basecalls performed using bcl2fastq version 2.20.0
RNA-seq and ChIP-seq reads were aligned to the mm10 genome using Hisat2 version 2.1.0. and bowtie2 version 2.3.3.1.
For RNA-seq analysis, To quantify uniquely aligned reads on respective annotated transcript loci (NCBI RefSeq transcripts), the htseq-count function with –s reverse option, part of the HTSeq package
For ChIP-seq analysis, Pearson correlation coefficient (R) in 1 kb bins between biological replicates were calculated with SeqMonk. Peak calling for ATAC- and ChIP-seq data were performed by MACS version 1.4.2 with default arguments; a cut-off P value of 10-25 was used. Relative ChIP-seq enrichments in respective peak regions were calculated by dividing input controls.
Genome_build: mm10
Supplementary_files_format_and_content: bam files were generated from each alignment step
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| Submission date |
Dec 17, 2019 |
| Last update date |
May 28, 2020 |
| Contact name |
Akihiko Sakashita |
| E-mail(s) |
[email protected]
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| Organization name |
Cincinnati Children's Hospital Medical Center
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| Street address |
3333 Burnet Ave.
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| City |
Cincinnati |
| State/province |
Ohio |
| ZIP/Postal code |
45229 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE142173 |
Endogenous retroviruses act as enhancers to drive species-specific germline transcriptomes in mammals |
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| Relations |
| BioSample |
SAMN13614433 |
| SRA |
SRX7396738 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4222000_Day3-Dox-minus-A-MYB-plus-2_mouse10_align_count_data.txt.gz |
97.8 Kb |
(ftp)(http) |
TXT |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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