 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Aug 30, 2019 |
| Title |
HA-DNMT3A2_ChIPseq_mESC_DNMT3A_KO |
| Sample type |
SRA |
| |
|
| Source name |
Mouse embryonic stem cells
|
| Organism |
Mus musculus |
| Characteristics |
chip antibody: HA (ThermoFisher, #88836)
cell line: ESC line V6.5 1A
cell type: C57BL/6 x 129S4/SvJae F1 embryo-derived embryonic stem cells
genotype: sgDnmt3a line stably expressing FLAG-HA tagged DNMT3A2 WT
|
| Growth protocol |
Mouse embryonic stem cells were maintained on gelatin-coated plates in Knockout DMEM (Gibco) supplemented with 15% ES-cell-qualified FBS (Gemini), 0.1 mM 2-mercapoethanol, 2mM L-glutamine (Life technologies) and LIF.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
To obtain a soluble chromatin extract, ~2x10^7 cells were resuspended in 1 mL LB1 (50 mM HEPES, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% NP-40, 0.25% Triton X-100, 1x Complete protease inhibitor) and incubated rotating at 4°C for 10 min. Samples were centrifuged, resuspended in 1 mL LB2 (10 mM Tris-HCl pH 8.0, 200 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 1x Compete protease inhibitor), and incubated rotating at 4°C for 10 min. Finally, samples were centrifuged, resuspended in 1 mL LB3 (10 mM Tris-HCl pH 8.0, 100 mM NaCl, 1 mM EDTA, 0.5 mM EGTA, 0.1% Na deoxycholate, 0.5% N-lauroylsarcosine, 1% Triton X-100, 1x Complete protease inhibitor), and homogenized by passing two times through a 27-gauge needle. Chromatin extracts were sonicated for 8 min (anti-HA ChIP) or 12 min (anti-histone PTM ChIP) using a Covaris E220 focused ultra-sonicator at peak power 140, duty factor 5, and cycles/burst 200. For histone PTM ChIP-Rx, after centrifugation soluble chromatin was spiked-in with soluble chromatin from Drosophila S2 cells that was similarly prepared and equivalent to 5-10% of the mouse/human cell chromatin. The lysates were incubated with 100 μl Pierce anti-HA beads (Themo Scientific, 88836) or with anti-H3K4me1 (Abcam, ab8895), anti-H3K9me3 (Abcam, ab8898), anti-H3K27ac (Active Motif, 39133), anti-H3K27me3 (Cell Signaling Tech, 9733), anti-H3K36me2 (Cell Signaling, 2901) or anti-H3K36me3 (Active Motif, 61101) antibody bound to 75 μl protein A or protein G Dnya1 magnetic beads (Invitrogen) and incubated overnight at 4°C with 5% kept as input DNA. Magnetic beads were washed with low salt buffer (150 mM NaCl; 0.1% SDS; 1% Triton X-100; 1 mM EDTA; 50 mM Tris-HCl), high salt buffer (500 mM NaCl; 0.1% SDS; 1% Triton X-100; 1 mM EDTA; 50 mM Tris-HCl), LiCl buffer (150 mM LiCl; 0.5% Na deoxycholate; 0.1% SDS; 1% Nonidet P-40; 1 mM EDTA; 50 mM Tris-HCl) and TE buffer (1 mM EDTA; 10 mM Tris-HCl). For ChIP-seq, beads were resuspended in elution buffer (1% SDS, 50 mM Tris-HCl pH 8.0, 10mM EDTA, 200 mM NaCl) and incubated for 30 min at 65°C. After centrifugation the eluate was reverse cross-linked overnight at 65°C. The eluate was then treated with RNaseA for 1 hr at 37°C and with Proteinase K (Roche) for 1 hr at 55°C and DNA was recovered using Qiagen PCR purification kit.
KAPA Hyper Prep Kit
|
| |
|
| Library strategy |
ChIP-Seq |
| Library source |
genomic |
| Library selection |
ChIP |
| Instrument model |
Illumina HiSeq 2500 |
| |
|
| Description |
According to manufacturer's protocol
|
| Data processing |
Raw ChIP sequencing reads were aligned using BWA version 0.7.17 with default parameters
Raw RNA sequencing reads were aligned using STAR version 2.5.3a with default parameters
Raw reads from whole-genome bisulfite sequencing were aligned using BWA (version 0.6.1). Low-quality sequence at the 3′ ends were trimmed. After alignment, we filtered duplicated or poorly mapping reads (>2% mismatches or aberrant insert size). Samtools (version 0.1.18) in mpileup mode was applied to call CpG methylation. We filtered CpGs with less than 5x coverage, overlapping with SNPs from dbSNPs (build 137) or located within the ENCODE DAC blacklisted regions or Duke excluded regions.
Genome_build: hg19, mm10 and dm6 for human, mouse and drosophila data, respectively
Supplementary_files_format_and_content: bigWig
|
| |
|
| Submission date |
May 16, 2019 |
| Last update date |
Aug 30, 2019 |
| Contact name |
Carmen Rivas |
| E-mail(s) |
mcarmen.rivas@usc.es
|
| Organization name |
Universidad de Santiago de Compostela
|
| Department |
CIMUS
|
| Lab |
P2L7
|
| Street address |
Avda Barcelona
|
| City |
Santiago de Compostela |
| ZIP/Postal code |
15706 |
| Country |
Spain |
| |
|
| Platform ID |
GPL17021 |
| Series (1) |
| GSE118785 |
H3K36me2 recruits DNMT3A and shapes intergenic DNA methylation landscapes |
|
| Relations |
| BioSample |
SAMN11663726 |
| SRA |
SRX5850005 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM3772692_mESC_sgDNMT3A_DNMT3A.bw |
230.2 Mb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
