Project description:We constructed a small RNA cDNA library, using small RNA fraction with a length of 19-29 bases, and we performed deep sequencing of the cDNA library.
Project description:Deep sequencing datasets are used for gRNA library analysis for large-scale screens. Using a lentiviral library that targets sequences across back-splicing sites of highly expressed human circRNAs, we show that a group of circRNAs are important for cell growth mostly in a cell-type specific manner and that a common oncogenic circRNA, circFAM120A, promotes cell proliferation in vitro and in vivo.
Project description:We performed deep sequencing of small RNA from mouse insulinoma (MIN6) cells or mouse liver using different library prepartion methods.
Project description:Test whether it is possible to conjugate a whole plasmid library into a recipient strain without loss of fidelity (as judged by aCGH analysis)
Project description:Data-independent acquisition (DIA) mass spectrometry (MS) data acquisition and targeted data extraction has become a promising strategy with enhanced identification coverage and consistent quantitation across multiple samples. However, coverage at the genome-depth still awaits to be improved. Here, we established a high quality proteome reference library from five lung cancer cell lines varying in epidermal growth factor (EGFR) mutation status and twenty two pooled patient-derived lung tumor tissues samples. By using high pH reverse phase (HpRP) fractionation, we were able to achieve 12,344 protein groups (223,091 unique peptide sequences, 344,430 precursors) from 191 data dependent acquisition (DDA) raw files acquired over similar chromatographic and Orbitrap Fusion Lumos MS platform, at 1% PSM and protein level false discovery rate (FDR). Application to tissue and cell line derived peptides also showed deep profiling of library based DIA. The established spectra library provides a useful resource for deep quantitative sensitive proteome profiling of clinical samples.
2021-03-05 | PXD019916 | JPOST Repository
Project description:Comparison of plasmid library culture methods
Project description:Recent studies highlight the importance of translational control in determining protein abundance, underscoring the value of measuring gene expression at the level of translation. We present a protocol for genome-wide, quantitative analysis of in vivo translation by deep sequencing. This ribosome profiling approach maps the exact positions of ribosomes on transcripts by nuclease footprinting. The nuclease-protected mRNA fragments are converted into a DNA library suitable for deep sequencing using a strategy that minimizes bias. The abundance of different footprint fragments in deep sequencing data reports on the amount of translation of a gene. Additionally, footprints reveal the exact regions of the transcriptome that are translated. To better define translated reading frames, we describe an adaptation that reveals the sites of translation initiation by pre-treating cells with harringtonine to immobilize initiating ribosomes. The protocol we describe requires 5 - 7 days to generate a completed ribosome profiling sequencing library. Ribosome profiling in cultured mammalian cells under three different footprinting conditions
Project description:Chromatin immunoprecipitation followed by ultra-high throughput (UHTP) sequencing (ChIP-seq) is a powerful tool to establish protein-DNA interactions genome-wide, and the primary limitation of its broad application at present is the often-limited access to sequencers. Here, we report a protocol, Mab-Seq, to generate preliminary quality evaluations and single-chromosome data for deep-sequencing libraries. We show that commercially available genomic microarrays can be used to maximize the efficiency of library creation, quickly generate preliminary data on a chromosomal scale, and help establish the depth to which novel libraries will require deep sequencing.
Project description:To evaluate the effect of mouse Siglec1 on B16F10 melanoma we used co-culture methos where HEK293T cells were transiently transfected with plasmid containg mouse Siglec1 or empty plasmid (mock). These HEK293T cells were cultured with B16F10-GFP mouse melanoma cells for 18 hours in low binding plates. After 18 hours of incubation on 37°C, 5% CO2, melanoma cells were FACS sorted based on GFP expression and total RNA was isolated and used for TruSeq RNA library construction for transcriptome analysis. Samples were from 3 independent experiments. The library preparation and NGS data aquisition were performed by Macrogen (Macrogen, Inc., Korea)
