|
| Status |
Public on Dec 01, 2012 |
| Title |
DNMT1 RIPSeq HL60 |
| Sample type |
SRA |
| |
|
| Source name |
HL60 Cell lines
|
| Organism |
Homo sapiens |
| Characteristics |
cell line: HL60
rip antibody: DNMT1
vendor: Abcam
catalog#: ab87656
|
| Treatment protocol |
RNA immunoprecipitated with DNMT1 antibody (Abcam cat# ab87656) and IgG (Sigma Aldrich) was processed for sequencing as described with some modifications. Double stranded cDNA was synthesized using the Just cDNA Double-Stranded cDNA Synthesis Kit (Agilent Technology, Santa Clara, CA) according to the manufacturer's instructions. Illumina sequencing libraries were constructed from these cDNA using ChIP-Seq sample preparation kit (cat# IP-102-1001, Illumina, San Diego, CA) with some modifications. Illumina paired-end adaptor and PCR primers were used to replace the single read adaptor and primers in the kit. Constructed libraries were subjected to a final size-selection step on a 10% Novex TBE Gels (Invitrogen, Carlsbad). DNA fragments of 175-200 bp were excised from SYBR-green-stained gel. DNA was recovered from the gel and quantified following Illumina's qPCR quantification protocol. A paired end sequencing of these libraries was then performed on Illumina GA IIx to achieve 2-76 bp reads.
|
| Growth protocol |
HL-60 were grown in DMEM supplemented with 10%FBS, at 37 °C in a humidified atmosphere with 5% CO2
|
| Extracted molecule |
total RNA |
| Extraction protocol |
DNMT1 interacting RNA was immunoprecipitated by DNMT1 antibody. RNA was reverse transcribed into ds-cDNA before sequencing library preparation using Illumina ChIP-Seq sample preparation kit (cat# IP-102-1001) following manufacturer's instruction. DNA at the length of 150-200 bp was recovered by gel size selection before sequencing.
|
| |
|
| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina Genome Analyzer IIx |
| |
|
| Description |
RNA IP against DNMT1
|
| Data processing |
Alignment: Paired-End reads were trimmed to 50bp and then aligned to the reference genome hg19 using BWA with the following parameters: bwa aln -o 1 -l 25 -k 2; bwa sampe -o 200.
The wig files were generated using MACS 1.3.7.1 with a resolution of 100bp.
|
| |
|
| Submission date |
Sep 15, 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, Discipline of Genetics
|
| 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) |
| GSE32162 |
RIPSEQ DNMT1 HL60 |
| GSE32260 |
Relationship between DNMT1-RNA interactions, DNA methylation and gene expression |
|
| Relations |
| SRA |
SRX103266 |
| BioSample |
SAMN00744402 |
