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| Status |
Public on Jul 24, 2024 |
| Title |
mESC, Flag-MBD5, CLIP, rep2 |
| Sample type |
SRA |
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| Source name |
embryos
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| Organism |
Mus musculus |
| Characteristics |
tissue: embryos
cell line: mESC
cell type: mouse embronic stem cell
genotype: Flag tagged MBD5
antibody: Flag (Abcam, #ab205606)
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| Treatment protocol |
Flag-MBD5 or Flag-MBD6 stably overexpression cell lines were constructed as follows: the coding sequence for human MBD5 or MBD6 were inserted into pPB-3XFlag-Hyg vector, which is a gift from Dr. Jianlong Wang lab at Columbia University. The expression plasmid was delivered into WT mESCs by lentivirus. The resulting cell lines were kept in mESC medium supplemented with 100 mg/L Hygromycin B (Gibco, 10687010).
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| Growth protocol |
All mESCs were kept in DMEM (Gibco, 11995065) supplemented with 15% Stem Cell Qualified Fetal Bovine Serum, Heat Inactivated (Gemini Bio Products, 100-525), 1 × L-glutamine (Gibco, 25030081), NEAA (Gibco, 25030081), LIF (MilliporeSigma, ESG1107), 1 × β-mercaptoethanol (Gibco, 21985023), 3 μM CHIR99021 (STEMCELL Technologies, 72052) and 1 μM PD0325901 (STEMCELL Technologies, 72182) at 37 °C and 5% CO2. The medium was replaced every day. ES cells were passaged on gelatin-coated plates twice to clear feeder cells before experiments.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Pellets were lysed by rotating at 4 °C for 15 minutes after passing through a 26 G needle (BD Biosciences). Embryo suspensions were sonicated on a bioruptor (Diagenode) with 30 s on/30 s off for 5 cycles. Lysates were cleared by centrifugation at 21,000 g for 15 minutes at 4 °C on a benchtop centrifuge. Supernatants were applied to MBD6 antibody (LSbio, LS-C158106-400) conjugated protein A beads (Invitrogen, 1001D) and left overnight at 4 °C on an end-to-end rotor. Beads were washed extensively with 1 ml wash buffer (50 mM HEPES pH 7.5, 300 mM KCl, 0.05% (v/v) NP-40, 1 × Halt™ Protease and Phosphatase Inhibitor Cocktail, 1 × RNaseOUT Recombinant Ribonuclease Inhibitor) at 4 °C for 5 times. Protein-RNA complex conjugated to the beads were treated by 8 U/μL RNase T1 (Thermo Scientific, EN0541) at 22 °C for 10 minutes with shaking. Input samples are digested in parallel. Then input and IP samples were separated on an SDS-PAGE gel and gel slices at corresponding size ranges were treated by proteinase K (Invitrogen, 25530049) elution. RNA was recovered with TRIZol reagent (Invitrogen, 15596026). Then T4 PNK (Thermo Scientific, EK0031) end repair was performed.
Library constructions were performed with NEBNext® Small RNA Library Prep Set for Illumina® (NEB, E7330S)
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| Library strategy |
RIP-Seq |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
Low quality reads were filtered using ‘fastq_quality_filter’, and adapter were clipped using ‘fastx_clipper’, then adapter-free were collapsed to remove PCR duplicates by using ‘fastx_collapser’ and finally reads longer than 15 nt were retained for further analysis (http://hannonlab.cshl.edu/fastx_toolkit/).
Reads from rRNA were removed. The preprocessed reads were mapped to mouse genome (mm10) using bowtie (version 1.0.0) with ‘-v 3 -m 10 -k 1 –best –strata’ parameters.
Mapped reads were separated by strands with samtools (version 1.16.1) and peaks on each strand were called using MACS2 (version 2) with parameter ‘-nomodel, --keep-dup 5, -g 1.3e8, -extsize 150’ separately. Significant peaks with q < 0.01 identified by MACS2 were considered.
Assembly: mm10
Supplementary files format and content: narrowPeaks (excecp for input samples)
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| Submission date |
Aug 21, 2023 |
| Last update date |
Jul 24, 2024 |
| Contact name |
Xiaoyang Dou |
| E-mail(s) |
xiaoyang.dou@sibcb.ac.cn
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| Organization name |
CAS Center for Excellence in Molecular Cell Science, CAS
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| Street address |
320 Yue Yang Road
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| City |
Shanghai |
| ZIP/Postal code |
200031 |
| Country |
China |
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| Platform ID |
GPL24247 |
| Series (2) |
| GSE241345 |
TET2 regulates histone H2AK119ub and leukemogenesis through LTR RNA m5C oxidation [seq_RNA-CLIP] |
| GSE241347 |
Chromatin-associated LTR RNA m5C oxidation by TET2 regulates leukemogenesis |
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| Relations |
| BioSample |
SAMN37096550 |
| SRA |
SRX21437452 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM7727532_mESC_Flag-MBD5_CLIP_rep2_peaks.narrowPeak.gz |
111.5 Kb |
(ftp)(http) |
NARROWPEAK |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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