Project description:Human PDCD4 wild-type (wt) promoter fragments were amplified from U2OS genomic DNA and X-box deletion mutants (mut) were generated using the QuikChange site-directed mutagenesis kit (Agilent Technologies). DNA probes for affinity purification with the same sequences were obtained by PCR using a biotinylated primer for labeling the 3' end (Thermo Fisher Scientific). DNA affinity purification with nuclear extracts from Nutlin-3a treated U2OS cells.
Project description:To identify the protein partners of SARS-CoV-2 -1 PRF RNA, we performed RNA pull-down assays using streptavidin magnetic beads. To obtain more accurate and reliable data, tRSA sequence-tagged -1 PRF and -1 FSE RNA probes were both used to capture binding proteins in our screen. As a random sequence control for nonspecificity, the equivalent proteins extracted from H1299 cells were incubated with tRSA-labeled -1 PRF and -1 FSE RNA probes. A high-throughput technology, LC/MS-MS(MS), was applied to identify the candidate interacting proteins of SARS-CoV-2 -1 PRF RNA.
Project description:We applied the solution hybrid selection approach to the enrichment of CpG islands (CGIs) and promoter sequences from the human genome for targeted high-throughput bisulfite sequencing. A single lane of Illumina sequences allowed accurate and quantitative analysis of 1 million CpGs in more than 21,408 CGIs and 15,946 transcriptional regulatory regions. More than 85% of capture probes successfully yielded quantitative DNA methylation information of targeted regions. In this study, we generated genome-wide, single-base resolution DNA methylation maps in three of the most commonly used breast cancer cell lines.Differentially methylated regions (DMRs) were identified in the 5?-end regulatory regions, as well as the intra- and intergenic regions, particularly in the X chromosome among the three cell lines. The single CpG resolution methylation maps of many known tumor suppressor genes were also established in the three cell lines. Here we present a novel approach that combines solution-phase hybrid selection and massively parallel bisulfite sequencing to profile DNA methylation in targeted CGI and promoter regions. We designed 51,466 single strand DNA oligonucleotides (160-mer) which target 23,441 CGIs and the transcription start sites of 19,369 known genes in the human genome. The synthetic long DNA oligonucleotides were converted into biotinylated RNA probes for solution-phase hybridization capture of target DNA. The captured genomic DNA was treated with sodium bisulfite, amplified by PCR and sequenced using Illumina GA IIx sequencer.