Project description:Forum domains are stretches of chromosomal DNA that are excised from eukaryotic chromosomes during their spontaneous non-random fragmentation. Mostly forum domains are of 50-200 kb in length, although larger domains, up to 500 - 700 kb, are also observed. We performed a genome-wide mapping of forum domains termini in human HEK293T cells cultured cells using deep sequencing of the termini. We found that forum domains termini correspond to the fragile sites in human chromosomes and forum domains contain clusters of several or many genes inside. The largest forum domains correspond to the coordinately expressed main clusters of HOX genes genes. Our results indicate that forum domains correspond to big multi-gene chromosomal units some of which could be co-coordinately activated or repressed. 2 sample examined: forum termini from HEK293T cells in two independent experiments
Project description:Large DNA fragments (mainly of 50-250 kb, denoted as forum domains) were isolated inside agarose plugs as described below in the extract protocol section. After ligation of 5'-bio-oligos to the blunt DNA termini the digestion with Sau3A enzyme was performed followed by selection of genome-wide preparation of forum domains termini on streptavidin-paramegnetic particles. After ligation of Sau3A adapter the termini were PCR amplified and used for deep sequencing.
Project description:Forum domains are stretches of chromosomal DNA that are excised from eukaryotic chromosomes during their spontaneous non-random fragmentation. Mostly forum domains are of 50-200 kb in length, although larger domains, up to 500 - 700 kb, are also observed. We performed a genome-wide mapping of forum domains termini in human HEK293T cells cultured cells using deep sequencing of the termini. We found that forum domains termini correspond to the fragile sites in human chromosomes and forum domains contain clusters of several or many genes inside. The largest forum domains correspond to the coordinately expressed main clusters of HOX genes genes. Our results indicate that forum domains correspond to big multi-gene chromosomal units some of which could be co-coordinately activated or repressed.
Project description:Eukaryotic chromosomes are subjected to spontaneous fragmentation even under quick isolation of DNA in a solid phase by strong treatment with 0.1 M EDTA, 1% SDS and proteinase K (1 mg/ml). The long DNA fragments of excised chromosomal DNA were denoted as forum domains. Mostly forum domains are of 50-200 kb in length, although larger domains, up to 500 - 700 kb, are also observed. The domains are delimited by hot spots of double-strand breaks (DSBs). We performed a genome-wide mapping of DSBs in human HEK293T cells cultured cells using Illumina deep sequencing of the termini of forum domains. We found that in rDNA units the hot spots of DSBs are distributed non-randomly. Mostly they are located in IGS. Genome-wide mapping of DNA DSBs in HEK293T cells
Project description:Eukaryotic chromosomes are subjected to spontaneous fragmentation even under quick isolation of DNA in a solid phase by strong treatment with 0.1 M EDTA, 1% SDS and proteinase K (1 mg/ml). The long DNA fragments of excised chromosomal DNA were denoted as forum domains. Mostly forum domains are of 50-200 kb in length, although larger domains, up to 500 - 700 kb, are also observed. The domains are delimited by hot spots of double-strand breaks (DSBs). We performed a genome-wide mapping of DSBs in human HEK293T cells cultured cells using Illumina deep sequencing of the termini of forum domains. We found that in rDNA units the hot spots of DSBs are distributed non-randomly. Mostly they are located in IGS.
Project description:Solanum lycopersicum RNA degradome sequencing Isolated polyadenylated RNA from total RNA extracts of Solanum lycopersicum, were ligated to 5'-adapter that includes an MmeI recognition site. The ligated products were purified again, reverse transcribed and cleaved with MmeI. The 5' fragments were purified from gel and ligated to a 3'- dsDNA adapter and PCR amplified. After PCR amplification the sample was subjected to Solexa/Illumina high throughput pyrosequencing. Please see www.illumina.com for details of the sequencing technology.
Project description:Vitis vinifera RNA degradome Isolated polyadenylated RNA from total RNA extracts of Vitis vinifera leaves, were ligated to 5'-adapter that include san MmeI recognition site. The ligated products were purified again, reverse transcribed and cleaved with MmeI. The 5' fragments were purified from gel and to a 3'- dsDNA adapter and PCR amplified. After PCR amplification the sample was subjected to Solexa/Illumina high throughput pyrosequencing. Please see www.illumina.com for details of the sequencing technology.
Project description:High-throughput sequencing of Arabidopsis thaliana endogenous small RNAs by 454 pyrosequencing. Keywords: high-throughput sequencing
Project description:To test if LEDGF/p75 influences distribution of Maedi-visna virus (MVV) integration sites, we infected human HEK293T, LKO (PSIP1-null), and LHKO (PSIP1/HDGFL2-null) cells with MVV-derived vector. Genomic DNA was isolated from infected cells, and chromosomal junctions at integrated U5 vDNA ends were amplified using linker-mediated PCR, sequenced using Illumina technology and mapped to human genome.
Project description:To investigate functional stress responsive mRNA isoform in human and mice, we performed high-throughput RNA sequencing (RNA-seq) of HEK293T and N2a cells under ER and metabolic stresses.
