 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Jan 17, 2020 |
| Title |
Mettl3_Control_mNET_r2 |
| Sample type |
SRA |
| |
|
| Source name |
Embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
cell type: mouse embryonic stem cells
strain: C57BL/6
antibody: mouse anti-RNA Polymerase II
|
| Treatment protocol |
The cells were lysed with ice-cold 4 ml of HLB/NP40 buffer (10 mM Tris-Hcl, pH 7.5, 10 mM NaCl, 0.5% NP40, and 2.5 mM MgCl2) and incubated on ice for 5 min. After the incubation, 1 ml of ice-cold HLB/NP40/Sucrose buffer (10 mM Tris-HCl pH 7.5, 10 mM NaCl, 0.5% NP40, 2.5 mM MgCl2, and 10% Sucrose) was under-laid and then the nuclei were collected under 1,400 rpm centrifuge at 4 oC for 5 min. Isolated nuclei were resuspended in 125 ml of NUN1 solution (20 mM Tri-HCl, pH 8.0, 75 mM NaCl, 0.5 mM EDTA, 50% Glycerol and proteinase inhibitor 1xComplete (Roche) followed by 1.2 ml NUN2 buffer (20 mM HEPES-KOH pH 7.6, 7.5 mM MgCl2, 0.2 mM EDTA, 300 mM NaCl, 1 M Urea, 1% NP40, proteinase inhibitor 1xComplete and phosphatase inhibitor 1xPhosStop [Roche]). 15 min incubation was carried out on ice with mixing by max speed vortex for 5 s every 4 min and then chromatin pellets were precipitated under 13,000 rpm centrifuge at 4 oC for 10 min.
|
| Growth protocol |
Cells were maintained in DMEM (Invitrogen) supplemented with 15% FBS, 1% nucleosides (100×),1 mM L-glutamine,1% nonessential amino acid, 0.1 mM 2-mercaptoethanol, 1,000 U/ml LIF, 3 μM CHIR99021 and 1 μM PD0325901 37 °C in 5% CO2.
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Isolated chromatin was washed in 100 l of 1x Micrococcal Nuclease (MNase) buffer (NEB) and then incubated with MNase (40 u/ml) on Thermomixer (eppendorf, 1,400 rpm) at 37 oC for 90 s. In order to inactivate MNase, EGTA (25 mM) was added immediately after the reaction and soluble-digested chromatin was collected by 13,000 rpm centrifuge for 5 min. The supernatant was diluted with 9 ml of NET-2 buffer (50 mM Tris-Hcl, pH 7.4, 150 mM NaCl, 0.05% NP40) and Pol II antibody-conjugated beads were added. 40 g of Pol II antibody was used for each mNET-seq experiment. Immunoprecipitation was performed at 4 oC for 1 hr. The beads were washed with 1ml of NET-2 buffer six times and with 500 l of 1xPNKT (1xPNK buffer and 0.1% Triton X-100) buffer once in the cold room. The washed beads were incubated in 100 l of PNK reaction mix (1xPNKT, 1 mM ATP and 0.05 U/ml T4 PNK 3’ phosphatase minus (NEB) on Thermomixer (1,400 rpm) at 37 oC for 6 min. After, the reaction beads were washed with 1ml of NET-2 buffer once and RNA was extracted with Trizol reagent. RNA was resolved on 8% denaturing acrylamide 7M urea gels for size purification. 35–100 nt fragments were eluted from the gel using RNA elution buffer (1 M NaOAc and 1 mM EDTA) and RNA was precipitated in 75% Ethanol.
RNA libraries were prepared according to the manual of NEBNext Multiplex Small RNA Library Prep Set for Illumina (New England Biolabs).
|
| |
|
| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina HiSeq 4000 |
| |
|
| Data processing |
Basecalls performed using CASAVA
Raw reads were trimmed with Trimmomatic-0.38, then aligned to mouse genome and transcriptome (mm10, version M19, 2018-08-30) using HISAT (version 2.1.0) with ‘--rna-strandness F’ parameters. Annotation files (version M19, 2018-08-30, in gtf format) were downloaded from GENCODE database (https://www.gencodegenes.org/).
Reads on different regions of each GENCODE annotated gene were counted using featureCounts.
Escaping Index was calculated as sense reads around TSS (the interval [TSS-50, TSS+250]) devided by sense reads on genebody (the interval [TSS+500, TES]) (Takayuki Nojima et al., 2015, Cell ).
Genome_build: mm10
Supplementary_files_format_and_content: Escapping Index for each gene and each sample.
|
| |
|
| Submission date |
Nov 18, 2019 |
| Last update date |
Jan 17, 2020 |
| Contact name |
Xiaoyang Dou |
| E-mail(s) |
[email protected]
|
| Organization name |
Center for Excellence in Molecular Cell Science, CAS
|
| Street address |
320 Yue Yang Road
|
| City |
Shanghai |
| ZIP/Postal code |
200031 |
| Country |
China |
| |
|
| Platform ID |
GPL21103 |
| Series (2) |
| GSE133600 |
The RNA N6-methyladenosine regulates chromatin remodeling and transcription |
| GSE140547 |
The RNA N6-methyladenosine regulates chromatin remodeling and transcription [mESC_seq_RNA_mNET] |
|
| Relations |
| BioSample |
SAMN13319929 |
| SRA |
SRX7175360 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
|
|
|
|
 |
