|
| Status |
Public on Oct 01, 2012 |
| Title |
RRBS_Lips3_p13 |
| Sample type |
SRA |
| |
|
| Source name |
Lips3 Cell lines
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: Lips3
digestion: MspI
treatment: bisulfite
|
| Treatment protocol |
Genomic DNA was isolated from the cell lines. DNA was digested with MspI (NEB), a methylation-insensitive enzyme that cuts C’CGG. Digested DNA was size selected on a 4% NuSieve 3:1 Agarose gel (Lonza). For each sample, two slices containing DNA fragments of 40-120 bp and 120-220 bp, respectively, were excised from the unstained preparative portion of the gel. These two size fractions were kept apart throughout the procedure including the final sequencing. Pre-annealed Illumina adaptors containing 5’-methyl-cytosine instead of cytosine were ligated to size-selected MspI fragments. Adapter-ligated fragments were bisulfite-treated using the EZ DNA Methylation kit (Zymo Research, Orange, CA). The products were PCR amplified, size selected, and sequenced on the Illumina GA IIxat a reading length of 36 bp.
|
| Growth protocol |
hES, LiPS 1 and LiPS3 cells were grown in ES KO DMEM supplemented with 15% KSR serumn in presence of 5ng/ml FGF, at 37 °C in a humidified atmosphere with 5% CO2
K562 cells were grown in DMEM supplemented with 10% FBS , at 37 °C in a humidified atmosphere with 5% CO2
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
DNA was digested with MspI (NEB), a methylation-insensitive enzyme that cuts C’CGG. Digested DNA was size selected on a 4% NuSieve 3:1 Agarose gel (Lonza). For each sample, two slices containing DNA fragments of 40-120 bp and 120-220 bp, respectively, were excised from the unstained preparative portion of the gel. These two size fractions were kept apart throughout the procedure including the final sequencing. Pre-annealed Illumina adaptors containing 5’-methyl-cytosine instead of cytosine were ligated to size-selected MspI fragments. Adapter-ligated fragments were bisulfite-treated using the EZ DNA Methylation kit (Zymo Research, Orange, CA). The products were PCR amplified, size selected before subjecting to sequencing.
|
| |
|
| Library strategy |
Bisulfite-Seq |
| Library source |
genomic |
| Library selection |
Reduced Representation |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
Reduced Representation Bisulfite Sequencing of HL60
|
| Data processing |
Sequencing reads were mapped to the reference genome hg18 using RRBSmap allowing 2 mismatches and methylation scoring was performed using the GBSA software.
|
| |
|
| Submission date |
Oct 25, 2011 |
| Last update date |
May 15, 2019 |
| Contact name |
Touati Benoukraf |
| E-mail(s) |
tbenoukraf@mun.ca
|
| Phone |
+1 (709) 864-6671
|
| Organization name |
Memorial University of Newfoundland
|
| Department |
Faculty of Medicine, Division of BioMedical Sciences
|
| Street address |
Craig L. Dobbin Genetics Research Centre,
|
| City |
St. John's |
| State/province |
NL |
| ZIP/Postal code |
A1B 3V6 |
| Country |
Canada |
| |
|
| Platform ID |
GPL10999 |
| Series (2) |
| GSE33230 |
Genome wide DNA methylation analysis of leukemia and reprogrammed leukemia cells (sequencing) |
| GSE33231 |
Genome wide DNA methylation analysis of leukemia and reprogrammed leukemia cells |
|
| Relations |
| SRA |
SRX120145 |
| BioSample |
SAMN00790491 |
