|
| Status |
Public on Jan 06, 2012 |
| Title |
Control ES cells 96hr, 5hmC glucosyltransferase treated DNA-Seq |
| Sample type |
SRA |
| |
|
| Source name |
Control ES_96hr_5hmC glucosyltransferase treated DNA-Seq
|
| Organism |
Mus musculus |
| Characteristics |
cell line: E14Tg2a
cell type: embryonic stem cells
transfected with: control siRNA
passages: 20-35
|
| Treatment protocol |
Cells were lysed 96 h after transfection in Trizol reagent, and the genomic DNA were prepared according to manufacturer's instructions. For genome-wide mapping of 5hmC, the genomic DNA obtained from control KD and Tet1 KD cells were processed using the QUEST 5hmC detection kit (Zymo Research) according to manufacturer's instructions (5-hmc: 5-hydroxymethylcytosine).
|
| Growth protocol |
The cells were routinely maintained on gelatin-coated plates in the ESGRO complete plus clonal grade medium (Millipore), and were used at passage 20–35 for experiments. For siRNA transfections, mESCs were cultured on gelatin-coated plates in normal mESC medium.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
MspI-digested DNA from treated and untreated samples derived from control and Tet1 KD mESCs were size-selected (40–220 bp) and sequenced using Illumina GAII.
|
| |
|
| Library strategy |
MRE-Seq |
| Library source |
genomic |
| Library selection |
Restriction Digest |
| Instrument model |
Illumina Genome Analyzer II |
| |
|
| Description |
Sample 2
|
| Data processing |
mm8-ESC-Control-GT-summary.graph.variableStep.scaled.wig; genome build: mm8
mm8-ESC-Control-GT.bed; genome build: mm8
Sequenced 36-bp reads were aligned to the reference genome (mouse NCBI36/mm8 assembly) using Bowtie 0.12.2 (36). Those reads that mapped to unique genomic locations with at most two mismatches were retained. Roughly ∼50–65% of all reads were mapped to unique genomic locations. To increase the mapping efficiency, we trimmed one base from the 3′-end of the unmapped reads, aligned the residual 36-bp reads to the reference genome and retained those that mapped to unique genomic locations. This procedure was repeated until the residual reads were of length 18, and, in each iteration, reads that mapped to unique genomic locations were retained for further analysis. The reads that mapped to unique genomic locations but whose corresponding 5'- to 3'-sequence in the reference genome neither started with CGG nor ended with GGC were considered noise and discarded. The rationale for this is because the MspI cleaves the DNA containing its recognition site CCGG at the inner C position (C/CGG) (37), the DNA fragments that is sequenced must all start with CGG or end with GGC (complementary strand). Reads that start or end with anything other than CGG or GGC, respectively, are likely to be byproducts of DNA degradation or sequencing errors. The resulting mapped reads for the four samples (control KD treated, control KD untreated, Tet1 KD treated and Tet1 KD untreated) were normalized by total reads in each sample so that the number of reads mapping to a specific site is directly comparable across all four samples. *wig files contain normalized and scaled data in wiggle format.
A site was defined as a 5hmC site only if the normalized read count at this site in the control untreated sample is at least 1.5-fold higher than that in the control treated sample. A 5hmC site was defined to have reduced 5hmC levels in Tet1 KD mESCs only if the 5hmC levels in Tet1 KD cells is at least 1.5 fold less compared to that in Control KD cells.
|
| |
|
| Submission date |
Jan 05, 2012 |
| Last update date |
May 15, 2019 |
| Contact name |
Raja Jothi |
| E-mail(s) |
jothi@mail.nih.gov
|
| Organization name |
National Institutes of Health
|
| Department |
National Institute of Environmental Health Sciences
|
| Lab |
Systems Biology
|
| Street address |
111 TW Alexander Drive; A314
|
| City |
RTP |
| State/province |
NC |
| ZIP/Postal code |
27709 |
| Country |
USA |
| |
|
| Platform ID |
GPL9250 |
| Series (2) |
| GSE34267 |
Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity |
| GSE34887 |
Acute depletion of Tet1-dependent 5-hydroxymethylcytosine levels impairs LIF/Stat3 signaling and results in loss of embryonic stem cell identity [MRE-seq] |
|
| Relations |
| SRA |
SRX114593 |
| BioSample |
SAMN00769944 |
