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Sample GSM1530533

Query DataSets for GSM1530533

Status

Public on Nov 02, 2014

Title

WT_shTdg_Vc_E14_WG.MABseq

Sample type

SRA

Source name

mouse embryonic stem cells

Organism

Mus musculus

Characteristics

cell line: E14Tg2A
genotype: wild-type (WT)
lentiviral infection: Tdg knockdown (shTdg)
vitamin c treatment: 100 ug/ml, 96hrs
chip antibody: none

Treatment protocol

Stable Tdg knockdown was achieved using a lentiviral system obtained from the National Institutes of Health (NIH) AIDS Research and Reference Reagent Program (https://www.aidsreagent.org/). This system allows selection of infected cells with puromycin. The expression of short hairpin RNA (shRNA) targeting Tdg (5'-GCAAGGATCTGTCTAGTAA-3') or the control shRNA (5'-GTTCAGATGTGCGGCGAGT-3') is under the control of U6 promoter. To generate lentiviruses, three transducing vectors (pTY, pNHP and pHEF1a-VSVG) were cotransfected into 293T cells. The supernatant containing lentiviruses was harvested at 48 and 60 hrs after transfection and concentrated using a centrifugal filter (EMD Millipore, Amicon Ultra 100k). To generate stable knockdown cells, mESCs were infected with lentivirus in the 12-well plate and puromycin (2 µg/ml) was added to the medium for selecting infected cells. For vitamin C (Sigma, A8960) treatment, control and Tdg knockdown mouse ESCs were treated with 100 µg/ml of vitamin C for 60 or 96hrs.

Growth protocol

V6.5 (control and Tdg knockdown), E14Tg2A (control, Tdg knockdown and Tet1/2–/–), and J1 (Dnmt1/3a/3b–/–) mouse embryonic stem cell (mESC) lines were cultured in feeder-free gelatin-coated plates in Dulbecco’s Modified Eagle Medium (DMEM) (GIBCO, 11995) supplemented with 15% FBS (GIBCO), 2 mM L-glutamine (GIBCO), 0.1 mM 2-mercaptoethanol (Sigma), nonessential amino acids (GIBCO), 1,000 units/ml LIF (Millipore, ESG1107). The culture was passaged every 2-3 days using 0.05% Trypsin (GIBCO).

Extracted molecule

genomic DNA

Extraction protocol

For whole genome (WG) MAB-seq analysis, genomic DNA was extracted from mESCs using the DNeasy Blood & Tissue Kit (Qiagen 69504). To capture H3K4me1- or H3K27me3-enriched chromatin for genome-scale MAB-seq/BS-seq analysis, ~15 million cells were cross-linked with 1% formaldehyde for 10 minutes at room temperature followed by exposure to 0.125 M glycine. After two washes with cold PBS, cells were collected and stored at -80 °C before use. Chromatin were extracted and lysed sequentially with lysis buffer (LB3, 10 mM Tris-HCl, pH8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na-Deoxycholate, 0.5% N-lauroylsarcosine). Chromatin was sonicated using a microtip (Branson sonifier 450) until the DNA fragments were reduced to 200-1000 bp in length. 10 μg antibodies were immobilized with 100 μl Dynal protein-G beads (Invitrogen) for at least 6 hours. Immunoprecipitation was performed overnight at 4 °C with antibody-conjugated protein-G beads. DNA/protein complexes were washed with RIPA buffer (50 mM Hepes-KOH, pH7.6, 500 mM LiCl, 1 mM EDTA, 1% NP-40, 0.7% Na-Deoxycholate) for five times and reverse cross-linked at 65 °C overnight. The DNA was treated sequentially with RNase A and proteinase K, and purified by phenol/chloroform extraction and ethanol precipitation. Following antibodies were used in ChIP assays: anti-H3K4me1 (ab8895, Abcam) and anti-H3K27me3 (07-449, Millipore).
For genome-scale MAB-seq or BS-seq, 200-500 ng of ChIP DNA (H3K4me1- or H3K27me3-enriched) was first spiked-in with unmethylated lambda DNA (1:400), which was then repaired and ligated to methylated (5mC) custom adapters (forward 5’-ACACTCTTTCCCTACACGACGCTCTTCCGATC*T-3’; reverse 5’-/5Phos/GATCGGAAGAGCACACGTCTGAACTCCAGTC-3’; the asterisk denotes phosphorothioate bond) with the NEBNext Ultra DNA Library Prep kit from Illumina (NEB). Adaptor-ligated DNA was then purified with 1.2x AMPure XP beads (Beckman Coulter). For BS-seq, methylated-adpator-ligated DNA was directly subjected to bisulfite conversion using Qiagen EpiTect DNA Bisulfite Kit (Qiagen, 59104) per manufacturer’s instructions, except that the thermal cycle was repeated twice. For MAB-seq, methylated adpator-ligated DNA was treated by M.SssI (New England Biolabs, M0226M) in a 50-µL reaction for two rounds. In each round of treatment, DNA was first incubated with 1.0 unit/µL M.SssI methylase (New England Biolabs, M0226M) for 4 hours in 25-µL reaction [1.25 µL of 20 unit/µL M.SssI and 0.5 µL of 32 mM SAM (final concentration: 640 µM)], and additional 25-µL containing same concentration of M.SssI (1.0 unit/µL) and SAM (640 µM) was supplemented to treat DNA for another 8 hours (in 50-µL). Of note, the first round of M.SssI treatment was performed in Mg++-free reaction buffer [10 mM Tris-HCl (pH 8.0), 50 mM NaCl, 10 mM EDTA], while the second round was carried out with NEB buffer #2 [10 mM Tris-HCl (pH 7.9), 50 mM NaCl, 10 mM MgCl2, 1 mM DTT]. DNA was purified by sequential Phenol/Chloroform/Isoamyl Alcohol (PCI, 25:24:1) extraction and ethanol precipitation after each round of M.SssI treatment. M.SssI-treated, methylated adaptor-ligated DNA was then subjected to bisulfite conversion using the EpiTect DNA Bisulfite Kit (Qiagen) as described above. Bisulfite-treated DNA was pre-amplified for 5 cycles using KAPA HiFi Uracil+ HotStart ReadyMix (KAPA) with indexed and universal primers from NEBNext Multiplex Oligos for Illumina (Index Primers Set 1). The optimal PCR cycle numbers required to generate the final libraries were then determined by quantitative PCR. Final libraries were generated by scaled-up PCR reactions using the cycles determined above, and purified with 1.2x AMPure XP beads. For whole-genome MAB-seq analysis, 1 µg non-enriched genomic DNA was first spiked-in with unmethylated lambda DNA (1:400) and was then treated with M.SssI (1st round protocol). M.SssI-treated genomic DNA (in 50-µL) was fragmented to an average size of 300-400 bp with Covaris M220 (20% duty factor, 200 cycles per burst, 80 sec x 2) using microTUBE AFA Fiber Screw-Cap. Sheared DNA was purified (1.2x AMPure XP beads), end-repaired and ligated to methylated adapters as described for H3K4me1/H3K27me3-MAB-seq. Methylated-adaptor-ligated DNA was treated with M.SssI again (2nd round protocol) before bisulfite conversion (Qiagen EpiTect DNA Bisulfite Kit). Bisulfite-treated DNA was prepared for sequensing as described above.

Library strategy

Bisulfite-Seq

Library source

genomic

Library selection

RANDOM

Instrument model

Illumina NextSeq 500

Description

processed data file: shTdg_Vc_merged_WG.MABseq.txt
processed data file: mESC.5fC5caC.sites.mm9_pval0.00025_cov10_bedgraph.gz
processed data file: mESC.5fC5caC.sites.mm9_pval0.00025_cov5_bedgraph.gz
processed data file: mESC.MABseqRawSignal.mm9_cov5_bedgraph.gz
processed data file: mESC.coverage.mm9_cov5_bedgraph.gz

Data processing

Raw sequencing reads were first trimmed for low-quality bases and adaptor sequences using Trimmomatic (http://www.usadellab.org/cms/?page=trimmomatic). Data quality was then examined with FastQC (http://www.bioinformatics.babraham.ac.uk/projects/fastqc/). The trimmed reads were mapped against the mouse genome (mm9 build) with Bismark using the bowtie2 mode (http://www.bioinformatics.babraham.ac.uk/projects/bismark/). PCR duplicates were removed using the Picard program (http://picard.sourceforge.net/). Sequencing base quality was first recalibrated with BisSNP (http://epigenome.usc.edu/publicationdata/bissnp2011/) and CpGs with low quality were removed (minimum mapping quality [mmq] >=10 and minimum base quality [mbq] score >= 5). CpGs overlapping with cell-line-specific SNPs in the mouse genome (dbSNP build 132) were removed from downstream analysis with BisSNP. All programs were performed with default setting (unless otherwise specified). For MAB-seq analysis, raw signals were calculated as % of T/(C+T) at each called CpG. For BS-seq analysis, raw signals were calculated as % of C/(C+T) at each called CpG.

Submission date

Oct 22, 2014

Last update date

May 15, 2019

Contact name

Hao Wu

E-mail(s)

haowu7@gmail.com

Phone

617-713-8660

Organization name

Harvard Medical School/HHMI