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| Status |
Public on Jun 16, 2024 |
| Title |
mESC_hTet2Delta |
| Sample type |
SRA |
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| Source name |
mouse embryonic stem cells (mESCs)
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| Organism |
Mus musculus |
| Characteristics |
cell line: mouse embryonic stem cells (mESCs)
cell type: mouse embryonic stem cells (mESCs)
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| Treatment protocol |
TETs expression was induced by treatment of 1ug/ml Doxcyclin (Sigma) 24 or48 hours post 70% confluent cells.
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| Growth protocol |
MOLM13 cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum (FBS) and antibiotics in humidified atmosphere with 5% CO2 at 37C
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
RNA was harvested using Rneasy mini plus kit (Qiagen). 1.0 ug of total RNA was used for the construction of sequencing libraries.Purified genomic DNA (30 ng, spiked in with 5% of unmethylated lambda genomic DNA, Promega) was sheared to 200–500 bp using Covaris M220 (Covaris, MA).
RNA libraries for RNA-seq were prepared using NEBNext Ultra Directional RNA Library Prep Kit (NEB #7760) following manufacturer's protocols. WGBS: The shared genomic DNA were then performed subjected t using EZ DNA methylation-lightning kit (Zymo Research) according to manufacturer’s instructions. The bisulfite treated genomic DNA were further proceeded to library preparation with xGenTM Methyl Seq DNA Library Prep Kit (IDT, CA) following the manufactures’ instructions. DamID:1 µg genomic DNA from mESCs expressing DamID only, TET2CD-Dam or TET2CDΔLCI-Dam is extracted and digested with DpnI (NEB,R0176) for 12 hrs at 37 °C. The digested DNA was purified by QIAquick PCR Purification Kit (Qiagen) to exclude uncut genomic DNA. Adaptors are ligated to the DpnI cutting fragments for PCR amplification. After digested with DpnII (NEB,R0543), DNA was then used as template for a PCR reaction to amplify the methylated fragments. DNA was then purified and sonicated into 200-300 bp for end repair, 3’ end adenylation and sequencing adaptor ligation. The enriched libraries are pooled and subjected to high-throughput sequencing. Micro-C:Micro-C libraries were prepared with Micro-C Kit (Dovetail, CA) following the manufacturer’s instructions. In brief, 1 million cells were resuspended and fixed by freshly made DSG (3 mM) for 15 min and then formaldehyde (1%) for another 10 min at room temperature. The crosslinking reaction was quenched with Tris buffer (pH = 7.5) to the final 0.75 M at room temperature. Fixed cells were washed twice and then subjected to chromatin fragmentation via MNase digestion for 15 min. After end repairing and proximity ligation, DNA were purified with streptavidin beads and adaptor ligation was performed on beads. An optimal PCR cycle for final library amplification was determined by quantification PCR and libraries were pooled and sequenced on Illumina Nova-seq platform with paired-end, 150/8/8/150 mode.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina NovaSeq 6000 |
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| Data processing |
RNA-seq data were mapped to the hg19 or mm10 genome assembly using STAR (version=2.7.10a) with default parameters. DESeq2 was used to call significantly differentially expressed genes (DEGs; log2 fold change >= 1 or <= -1, q-value < = 0.05) between MOLM13 expression TET2CD and TET2CDΔLCI.
Raw WGBS fastq files were mapped to the hg19/mm10 genome assembly using the bsmap-2.89 software with “-v 6 -n 1 -q 3 -r 1” parameters. The bisulfite conversion ratios were estimated using unmethylated lambda DNA. Mcall modual in MOABS (56) was used to call the mCG/CG ratios for each CpG site. Mcomp modual was used to call DMRs with parameter “–minNominalDif = 0.2–minDmcsInDmr 3–maxDistConsDmcs 500”. The CpGs with coverage > = 5 was used for downstream analysis.
Raw DamID fastq data were aligned to mm10 genome assembly using bowtie2.4.5 with default parameters. Mapped DamID data were analyzed by damidseq_pipeline (55). Specifically, sequencing fragments located on GATC sites were counted, normalized and bedGraph file with DamID-fusion / DamID-only log2ratio were generated. Find_peaks perl script in damidseq_pipeline was used to call significant peaks. The genome was splitted into 5k bins and the mean signals of mESC Flag-Tet2 ChIP, Dam-fusion with Tet2CD/Dam-only ratios, mESC H3K27ac and mESC H3K9me3 on each 5k bin were calculated. The pearson correlation coefficient among them were calculated and plotted. IGV was used to visualize the tracks.
Raw CMS-IP fastq data were mapped to the mm10/hg19 genome assembly using bsmap-2.89 with default parameters. After duplication removal, CMS peaks were called by using macs2 with default parameters. Bedtools merge was used to generate merged peaks for all samples. Reads numbers in each peak were counted if there was >1 bp overlap between reads location and peak region. The raw counts file with row as each peak, column as samples was used as input to DESeq2 and differentially significantly CMS peaks (q value < = 0.05) between the control and TET2CD or control and TET2CDΔLCI groups were called. Volcano plots were plotted using the R package ggplot2.
For Micro-C data analysis, we used Micro-C data analysis pipeline in https://micro-c.readthedocs.io/en/latest/ to process Micro-C data. Specifically, we used bwa-mem (0.7.17) algorithm to map raw fastqs on hg19 version genome with the following command: bwa mem -5SP -T0 -t16 hg38.fasta MicroC_2M_R1.fastq MicroC_2M_R2.fastq -o aligned.sam. PCR duplicates were removed. Pairtools pipeline was used to identify ligation event from mapping results and the validate pairs was extracted for downstream analysis. Juice box tools (57) was used to generate contact matrix (*.hic) data which can be visualize in Juicebox. runHiCpca.pl in Homer was used to calculate PCA1 value (A/B comparment) for 50k bins and the PCA1 value was corrected by using MOLM13 H3K27ac data (GSM2136938).
Assembly: mm10/hg19
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| Submission date |
Sep 17, 2022 |
| Last update date |
Jun 16, 2024 |
| Contact name |
Jia Li |
| E-mail(s) |
jiali@tamu.edu
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| Organization name |
Texas A&M U Health Science Center
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| Department |
CEDP
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| Lab |
Jia Li Lab
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| Street address |
2121 W HOLCOMBE BLVD
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| City |
Houston |
| State/province |
Texas |
| ZIP/Postal code |
77030 |
| Country |
USA |
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| Platform ID |
GPL24247 |
| Series (1) |
| GSE183934 |
Perturbation of TET2 condensation induces genome-wide promiscuous DNA hypomethylation and curtails leukemia cell growth |
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| Relations |
| BioSample |
SAMN30911447 |
| SRA |
SRX17617327 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM6589445_mESC_Tet2Delta.total.d5.bw |
206.3 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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