|
| Status |
Public on Dec 10, 2018 |
| Title |
HSC_6 OE Tr.2_methylation |
| Sample type |
genomic |
| |
|
| Source name |
Cord blood 6
|
| Organism |
Homo sapiens |
| Characteristics |
cell type: CD34+ HSCs
primary tissue: cord blood
dnmt3a transcript: DNMT3A transcript 2
|
| Treatment protocol |
For knockdown (KD) or overexpression (OE) of specific transcripts the HSPCs were lentivirally infected 1 day after isolation and selected with 2.5 µg/mL puromycin at day 2 post infection. To knockdown DNMT3A transcripts we designed short hairpin RNAs (shRNAs) to target transcript-specific exons: exon 5 of transcript 1+3 (ENSE00001486208), exon 2 of transcript 2 (ENSE00001486123), and exon 4 of transcript 4 (ENSE00001559474). In brief, forward and reverse oligonucleotides (Metabion, Planegg, Germany) were joined and ligated into the pLKO.1 vector (Addgene, Cambridge, MA, USA) that drives shRNA expression from the human U6 promoter and carries the puromycin resistance gene. For constitutive overexpression of DNMT3A transcripts the sequences of the transcripts were amplified from cDNA of human blood cells with the High-Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Waltham, MA, USA) using exon-specific primers.
|
| Growth protocol |
Cord blood samples were taken after written consent using guidelines approved by the Ethic Committee on the Use of Human Subjects at the University of Aachen (Permit number: EK 187-08). CD34+ HSPCs were isolated from cord blood and cultured in StemSpan Serum-Free Expansion Medium supplemented with 10 μg/mL heparin, 20 ng/mL thrombopoietin, 10 ng/mL stem cell factor, 10 ng/mL fibroblast growth factor 1 and 100 U/mL penicillin/streptomycin.
|
| Extracted molecule |
genomic DNA |
| Extraction protocol |
Genomic DNA was isolated using the NucleoSpin Tissue kit (Macherey-Nagel) acording to the manufacturers instructions.
|
| Label |
Cy5 and Cy3
|
| Label protocol |
Standard Infinium HD Methylation Assay protocol
|
| |
|
| Hybridization protocol |
Bisulphite converted DNA was amplified, fragmented and hybridised to Illumina Infinium Human Methylation450 Beadchip using standard Illumina protocol.
|
| Scan protocol |
Standard Infinium HD Methylation Assay protocol
|
| Description |
SAMPLE 22
overexpression
|
| Data processing |
Initial analysis was performed by the Genomestudio 2010.3 (Modul M Version 1.8.5). Data were normalized with internal controls according to Illumina´s standard procedures. Methylation level at each locus was calculated with the GenomeStudio Methylation module as beta-value (ranging from 0 to 1). The number of beads per feature varies between chips and beta-values were calculated as average of at least three technical replica.
|
| |
|
| Submission date |
Aug 23, 2017 |
| Last update date |
Dec 11, 2018 |
| Contact name |
Wolfgang Wagner |
| E-mail(s) |
wwagner@ukaachen.de
|
| Phone |
+49 241 8088611
|
| Organization name |
RWTH Aachen University
|
| Department |
Helmholtz Institute for Biomedical Engineering
|
| Lab |
Stem Cell Biology and Cellular Engineering
|
| Street address |
Pauwelsstrasse 20
|
| City |
Aachen |
| ZIP/Postal code |
52074 |
| Country |
Germany |
| |
|
| Platform ID |
GPL13534 |
| Series (2) |
| GSE103006 |
Variants of DNMT3A cause transcript-specific DNA methylation changes |
| GSE103008 |
Variants of DNMT3A cause transcript-specific DNA methylation and gene expression changes |
|
