Project description:Genome editing was conducted on a t(3;8) K562 model to investigate the effects of deleting different modules or CTCF binding sites within the MYC super-enhancer. To check mutations after targeting with CRISPR-Cas9 we performed amplicon sequencing using the Illumina PCR-based custom amplicon sequencing method using the TruSeq Custom Amplicon index kit (Illumina). The first PCR was performed using Q5 polymerase (NEB), the second nested PCR with KAPA HiFi HotStart Ready mix (Roche). Samples were sequenced paired-end (2x 250bp) on a MiSeq (Illumina).
Project description:Omental adipose tissue was obtained from 24 women aged 25-40 years with a healthy singleton pregnancy and a pre-pregnancy BMI of 18.5-26 kg/m2. Genomic DNA was extracted for promoter DNA methylation analysis of candidate genes LEP, TNF, NPY, POMC and SOCS3 using bisulfite amplicon sequencing.
Project description:The soybean (Glycine max) seed coat has distinctive, genetically programmed patterns of pigmentation and the recessive k1 mutation can epistatically overcome the dominant I and i-i alleles, which inhibit seed color by producing small interfering RNAs (siRNAs) targeting chalcone synthase (CHS) mRNAs. Small RNA sequencing of dissected regions of immature seed coats demonstrated that CHS siRNA levels cause the patterns produced by the i-i and i-k alleles of the I locus, which restrict pigment to the hilum or saddle region of the seed coat, respectively. To identify the K1 locus, we compared RNA-Seq data from dissected regions of two Clark isolines having similar saddle phenotypes mediated by CHS siRNAs but different genotypes (homozygous i-k K1 versus homozygous i-i k1). By examining differentially expressed genes, mapping information, and genome resequencing, we identified a 129-bp deletion in Glyma.11G190900 encoding Argonaute5 (AGO5), a member of the Argonaute family. Amplicon sequencing of several independent saddle pattern mutants from different genetic backgrounds revealed independent lesions affecting AGO5, thus establishing Glyma.11G190900 as the K1 locus. Non-functional AGO5 from k1 alleles leads to altered distributions of CHS siRNAs, thus explaining how the k1 mutation reverses the phenotype of the seed coat regions from yellow to pigmented, even in the presence of the normally dominant I or i-i alleles.
Project description:Whole-genome array comparative genomic hybridization (aCGH) of human ependymoma tumors. DOP-PCR products were spotted in triplicate onto NexterionTM Slide E epoxysilane-coated slides (PEQLAB, Erlangen, Germany) using a spotting robot (VersArray ChipWriterTM Pro system,BioRad, Munich, Germany) at 20C and 40% humidity. After spotting, slides were cross-linked,baked for 1 hr at 80C, and cross-linked again.
Project description:These vector oligonucleotide hybridizations were performed to determine the quality of the spotting process. EST with low signal values of the vector array were excluded from the analysis, since they probably were mis-amplified or mis-spotted Keywords: spotting control
Project description:Co-amplification at chromosomes 8p11-8p12 and 11q12-11q14 occurs often in breast tumors, suggesting possible cooperation between genes in these regions in oncogenesis. We used high resolution array comparative genomic hybridization (array CGH) to map the minimal amplified regions. The 8p and 11q amplicons are complex and consist of at least four amplicon cores at each site. Candidate genes mapping to these regions were identified by combining copy number and RNA and protein expression analyses. Funcational analysis for transformation was further carried out with candidate genes to determine candidate oncogenes.
Project description:Amplicon-based targeted re-sequencing analysis was performed in the patient-derived gliobastoma cell culture samples. For this purpose, genomic DNA (gDNA) was isolated and DNA libraries were prepared using the TruSeq Custom Amplicon Low Input (Illumina, Inc.) technology. By this, a pool of 375 amplicons was generated for each single sample in order to enrich for the target genes ATRX1, EGFR, IDH1, NF1, PDGFRA, PIK3CG, PIK3R1, PTEN, RB1 and TP53. Sequencing was performed on the Illumina MiSeq® next generation sequencing system (Illumina Inc.) and its 2 x 250 bp paired-end v2 read chemistry. The resulting reads were quality controlled and mapped against the human reference genome (hg19). For all samples, sequence variations of the amplified regions of interest in comparison to the human reference sequence were identified and filtered based on reliability.
