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GEO help: Mouse over screen elements for information. |
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| Status |
Public on Dec 11, 2018 |
| Title |
shRnf2 rep#2 (ATAC-seq) |
| Sample type |
SRA |
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| Source name |
mouse embryonic stem cell
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| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryonic stem cell
cell line: E14
knockdown: shRnf2
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| Treatment protocol |
pLKO lentiviruses, generated with Plat-E cells, were added to ESCs and medium was changed after 8 hours. Cells were selected with 2 μg/ml puromycin for 2 days, and harvested after a further 2 days.
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| Growth protocol |
Mouse ESCs were grown in serum+LIF medium: DMEM-HIGH GLUCOSE (SH30022.01, HyClone), 15% fetal bovine serum (S1580-500, Biowest), 1% L-Glutamine (35050-061, GIBCO), 1% sodium pyruvate (25-000-CIR, Corning), 1% non-essential amino acids (25-025-CI, Corning), 0.5% penicillin-streptomycin solution (SV30010, HyClone), 0.1 mM 2-mercaptoethanol (21985-023, GIBCO), 0.01% mLIF (ESG1107, Millipore).
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
ATAC-seq was performed as described (Buenrostro et al., 2015). In brief, a total of ~50,000 cells were washed once with 50 μl of cold PBS and resuspended in 50μl lysis buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2, 0.2% (v/v) IGEPAL CA-630). The suspension of nuclei was then centrifuged for 10 min at 500g at 4°C, followed by the addition of 50 μl transposition reaction mix (25 μl TD buffer, 2.5 μl Tn5 transposase and 22.5 μl nuclease-free H2O) of Nextera DNA library Preparation Kit (96 samples) (FC-121-1031, Illumina). Samples were then PCR amplified and incubated at 37°C for 30min. DNA was isolated using a MinElute Kit (Qiagen). ATAC-seq libraries were first subjected to 5 cycles of pre-amplification. To determine the suitable number of cycles required for the second round of PCR the library was assessed by quantitative PCR as described (Buenrostro et al., 2015), and the library was then PCR amplified for the appropriate number of cycles. Libraries were purified with a Qiaquick PCR (Qiagen) column and were quantified using a KAPA Library Quantification kit (KK4824, Kapa Biosystems) according to the manufacturer’s instructions. Library integrity was checked by gel electrophoresis. Finally, the ATAC library was sequenced on a NextSeq 500 using a NextSeq 500 High Output Kit v2 (150 cycles) (FC-404-2002, Illumina) according to the manufacturer’s instructions.
According to the extract protocol
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| Library strategy |
ATAC-seq |
| Library source |
genomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
bcl2fastq conversion software v2.17 used for basecalling.
Reads were aligned to the mouse (mm10) genome using bowtie2
Peaks were called using MACS2
Genome_build: mm10
Supplementary_files_format_and_content: Processed peak (BED) files and bigWig tracks
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| Submission date |
Dec 14, 2017 |
| Last update date |
Dec 12, 2018 |
| Contact name |
Jiangping He |
| E-mail(s) |
he_jiangping@grmh-gdl.cn
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| Organization name |
Guangzhou Institutes of Biomedicine and Health (GIBH), CAS
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| Street address |
Kai yuan avenue 190
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| City |
GuangZhou |
| ZIP/Postal code |
510530 |
| Country |
China |
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| Platform ID |
GPL19057 |
| Series (2) |
| GSE108086 |
Epigenetic control of genes and transposable elements [ATAC-seq] |
| GSE108091 |
Epigenetic control of genes and transposable elements |
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| Relations |
| BioSample |
SAMN08177857 |
| SRA |
SRX3471736 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM2889300_shRnf2.ATAC-Seq.rep2..bw |
253.4 Mb |
(ftp)(http) |
BW |
| GSM2889300_shRnf2.rep2.peaks.bed.gz |
345.0 Kb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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