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SLIC-CAGE of paired-end sequencing data in mouse islets

ABSTRACT: SLIC-CAGE of paired-end sequencing data in mouse islets

PROVIDER: PRJEB79386 | ENA |

REPOSITORIES: ENA

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SLIC-CAGE of paired-end sequencing data in mouse islets

Project description:Super-Low Input Carrier Cap Analysis of Gene Expression (SLIC-CAGE) to identify transcription start sites (TSS) and existing genome-wide maps of islet histone marks to characterise the contribution of transcriptional regulation of LKB1-mediated control of gene expression in mouse β-cells. SLIC-CAGE was performed as in (Cvetesic et al. - doi: 10.1101/gr.235937.118) using 100 ng of total RNA extracted as described above from islets isolated from a 18 week old female mouse on a C57BL6/J background. 12 cycles were performed for library amplification. The amplified library was purified with AMPure XP beads and visualised using a HS DNA chip (Bioanalyzer, Agilent). The library was sequenced on a HiSeq2500 instrument with paired-end, 150bp reads). SLIC-CAGE paired-end sequencing data was aligned to GENCODE assembly annotation version GRCm38.p6 using the STAR alignment tool v2.4.2a.

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SLIC-CAGE (Super-Low Input Carrier-CAGE) development: comparison with Saccharomyces cerevisiae nAnTi-CAGE and nanoCAGE libraries and validation of SLIC-CAGE on Mus musculus total RNA

Project description:We developed SLIC-CAGE (Super-Low Input Carrier-CAGE) approach to capture 5'end of RNA polymerase II transcripts from as little as 5-10 ng of total RNA. The dramatic increase in sensitivity compared to existing CAGE methods is achieved by specially designed, selectively degradable carrier RNA. We tested SLIC-CAGE on Saccharomyces cerevisiae (BY4741 strain) and produced libraries from 1-100 ng of total cellular RNA. We also produced S. cerevisiae nAnT-iCAGE libraries as the current gold-standard CAGE libraries using the recommended 5 micrograms of total cellular RNA to assess the quality of SLIC-CAGE libraries produces with up to 1000-fold less material. We provide a direct comparison between SLIC-CAGE and the latest nanoCAGE protocol (libraries created using S. cerevisiae total RNA) and show that SLIC-CAGE produces unbiased libraries of higher complexity and quality than nanoCAGE. Finally, we provide SLIC-CAGE libraries on mouse embryonic stem cells (E14) using 5-100 ng of total cellular RNA as starting material.

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