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| Status |
Public on Mar 28, 2012 |
| Title |
mis24_d12_MethylC-seq |
| Sample type |
SRA |
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| Source name |
H9 cells
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| Organism |
Homo sapiens |
| Characteristics |
days differentiated: 12 days
sirna: mismatch control #24
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| Treatment protocol |
In order to differentiate hESCs into neuroepithelium (NE), colonies were detached from feeders (day 0) with dispase (1 mg/mL) until the edges of the colonies began to lift. The clumps of cells were gently blown off and washed once with hESC medium without bFGF. The aggregates were cultured in hESC medium (without bFGF) for 4 days in Ultra Low Attachment flasks. Then, aggregates were grown in neural induction medium (DMEM/F12, N2 supplement, nonessential amino acids, 2 μg/ml heparin, and 10 ng/ml bFGF) for up to 3 additional days. On day 7, the aggregates were plated onto laminin-coated tissue culture plates.
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| Growth protocol |
H9 hESCs were grown feeder-free on Matrigel and transduced with either the DNMT3B KD or MIS virus. GFP-positive colonies were mechanically dissociated to isolate individual subclones. The individual subclones were on Matrigel in hESC medium (modified Eagle's medium/F-12 containing 20% KnockOut serum replacer, 0.1 mM nonessential amino acids, 1 mM L-glutamine, 0.1 mM beta-mercaptoethanol) that had been conditioned on mouse embryonic fibroblasts (MEFs) and then supplemented with 4 ng/ml bFGF
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| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA from H9 human embryonic stem cells was fragmented to ~300bp using Diagenode's Bioruptor. To create the sequencing libraries, 5 ug of DNA was end repaired using 1X T4 DNA ligase buffer, 400 uM dNTPs, 15 U T4 DNA polymerase (NEB), and 50 U PNK (NEB) for 30 minutes at 20 degrees Celsius. After PCR purifying (Qiagen), adenine bases were appended to the ends using 1X NEB 2 buffer, 200 uM dATP, and 15 U Klenow Fragment (3' to 5' exo-, NEB) for 30 minutes at 37 degrees Celsius. After another DNA purification using the PCR MinElute kit (Qiagen), 3 uL of Illumina's methylated sequencing adapters were ligated on using 1X ligase buffer and 5 uL Quick T4 DNA Ligase (NEB) for 30 minutes at room temperature. After a final PCR purification, 500 ng of library was bisulfite converted using Zymo's EZ DNA Methylation-Direct kit according to the manufacturer's instructions. The library was then amplified using 2.5 U PfuTurbo Cx Hotstart DNA Polymerase (Stratagene) for 12 cycles using Illumina's standard amplification protocol.
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| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
RANDOM |
| Instrument model |
Illumina HiSeq 2000 |
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| Description |
MethylC-seq
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| Data processing |
100 bp fastq reads were truncated to 76 bp and aligned to the genome (hg18) using BS-Seeker, allowing for up to two mismatches with the reference sequence. Custom perl scripts were used to calculate percent methylation for each genomic CpG site with read coverage. Methylation data for both DNA strands were combined. Only one read starting at a given genomic coordinate was kept to eliminate clonal replicates. Red=80-100% methylation, green=60-80%, blue=1-60%, black=0%
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| Submission date |
Sep 21, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Janine LaSalle |
| E-mail(s) |
jmlasalle@ucdavis.edu
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| Organization name |
UC Davis
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| Street address |
Medical Microbiology and Immunology
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| City |
Davis |
| State/province |
CA |
| ZIP/Postal code |
95616 |
| Country |
USA |
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| Platform ID |
GPL11154 |
| Series (1) |
| GSE32268 |
Role of DNMT3B in the regulation of early neural and neural crest specifiers |
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| Relations |
| SRA |
SRX098317 |
| BioSample |
SAMN00723064 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM799466_MIS24_d12_percentmeth_chr1.BED.gz |
8.4 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr10.BED.gz |
5.0 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr11.BED.gz |
4.9 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr12.BED.gz |
4.7 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr13.BED.gz |
2.8 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr14.BED.gz |
3.2 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr15.BED.gz |
3.1 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr16.BED.gz |
4.2 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr17.BED.gz |
4.5 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr18.BED.gz |
2.4 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr19.BED.gz |
4.3 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr2.BED.gz |
7.8 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr20.BED.gz |
2.8 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr21.BED.gz |
1.4 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr22.BED.gz |
2.3 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr3.BED.gz |
5.8 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr4.BED.gz |
5.0 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr5.BED.gz |
5.3 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr6.BED.gz |
5.3 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr7.BED.gz |
5.5 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr8.BED.gz |
4.7 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chr9.BED.gz |
4.3 Mb |
(ftp)(http) |
BED |
| GSM799466_MIS24_d12_percentmeth_chrX.BED.gz |
4.1 Mb |
(ftp)(http) |
BED |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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