Project description:Pull-down of endogenous N-terminally TAP-tagged ERBP1 (Tb927.10.14150). RNA of unbound and eluted samples was purified, rRNA of unbound sample was depleted, and RNA was analysed by RNA-Seq.
Project description:Coverage of short-read RNA-seq is highly non-uniform across transcripts even in genes with only one expressed isoform, contrary to biological expectation. We investigate the impact of several library preparation factors on the non-uniformity of coverage. Specifically, a mouse liver sample is prepared under varying ribosomal depletion (PolyA / rRNA digestion / rRNA pull-down / NoSelection), PCR ramp rate, and fragment length along with one heart sample without selection.
Project description:Analysis of the protein coding transcriptome by the RNA sequencing requires either enrichment of the desired fraction of coding transcripts or depletion of the abundant non-coding fraction consisting mainly of rRNA. We propose an alternative mRNA enrichment strategy based on the RNA-binding properties of the human IFIT1, an antiviral protein recognizing cap 0 RNA. Here, we compare for Saccharomyces cerevisiae an IFIT1-based mRNA pull-down with yeast targeted rRNA depletion by the RiboMinus method. IFIT1-based RNA capture depletes rRNA more effectively, producing high quality RNA-seq data with an excellent coverage of the protein coding transcriptome, while depleting cap-less transcripts such as mitochondrial or some non-coding RNAs. We propose IFIT1 as a cost effective and versatile tool to prepare mRNA libraries for a variety of organisms with cap 0 mRNA ends, including diverse plants, fungi and eukaryotic microbes.
Project description:We have recently found that the human methyltransferase ZCCHC4 is responsible for introducing the single m6A modification in 28S rRNA. The project is aimed at further elucidating the functional consequences of such methylation. To this end, protein mass spectrometry is utilized to identify proteins that bind preferentially to the methylated or unmethylated form of the m6A containing hairpin sequence in 28S rRNA. Specifically, this is done by incubating a HeLa cell extract with an methylated or unmethylated RNA oligonucleotide containing a biotin affinity tag, which is used to pull-down the RNA with associated proteins.
Project description:To re-evaluate FoxP3 sequence specificity, we performed an unbiased pull-down of genomic DNA with recombinant FoxP3 protein. Use of genomic DNA, as opposed to synthetic DNA oligos, enables testing sequence specificity in the context of naturally existing repertoire of sequences. De novo motif analysis showed a strong enrichment of TnG repeats (n=2-5) by FoxP3 pull-down, using either MBP alone pull-down or input as a control.
Project description:We employed chromatin pull-down and deep sequencing to globally identify HetR DNA targets in vivo at 6 hours after fixed-nitrogen deprivation. We identified novel DNA binding targets of tagged HetR-6xHis and defined a consensus HetR binding site from these HetR target sequences. Chromatin pull down of hetR mutant strain UHM103 carrying pAM4375, which expresses HetR-6xHis, and a wild-type control. One dataset was collected.
Project description:We expressed biotin-tagged transcription factor TFII-I (GTF2I) in K562 cells and subjected crosslinked chromatin to streptavidin mediated pull down. We subjected the isolated DNA to Illumina sequencing. We identified 18920 TFII-I binding peaks. Nearest gene analysis revealed 5886 genes associated with a TFII-I binding peak in proliferating K562 cells. We generated two K562 single cell clones stably expressing bio-tagged TFII-I, and K562 cells expressing only the BirA protein biotin ligase from E.coli. These three clones were subjected to streptavidin mediated pull down and Illumina sequencing.