Biology subjects

Shi, R.

Publications and source records attributed to Shi, R..

4 recordsLinked to original sources

bioRxiv2018

Filter paper-based spin column for low throughput nucleic acid purification

We describe a method of recharging used spin column or assembling homemade spin column using filter paper as binding material for low throughput nucleic acid purification. We evaluated the efficiency of filter paper based spin columns in the purification of different type of nucleic acids. For instance, by following protocols of respective commercial kits, we found that filter paper to be a useful binding material for purification of many types of nucleic acids, including plant genomic DNA, plant total RNA, PCR product, and DNA from agarose gels. We also found that filter paper has a weak binding affinity to plasmid DNA in tested miniprep protocols. Also, we present the protocols of using filter paper recharged spin column or homemade spin column for low throughput purification of plant genomic DNA and plant total RNA with commercial kit buffer leftover and less expensive homemade buffer.

molecular biology↗

bioRxiv2018

Two-dimensional local Fourier image reconstruction via domain decomposition Fourier continuation method

The MRI image is obtained in the spatial domain from the given Fourier coefficients in the frequency domain. It is costly to obtain the high resolution image because it requires higher frequency Fourier data while the lower frequency Fourier data is less costly and effective if the image is smooth. However, the Gibbs ringing, if existent, prevails with the lower frequency Fourier data. We propose an efficient and accurate local reconstruction method with the lower frequency Fourier data that yields sharp image profile near the local edge. The proposed method utilizes only the small number of image data in the local area. Thus the method is efficient. Furthermore the method is accurate because it minimizes the global effects on the reconstruction near the weak edges shown in many other global methods for which all the image data is used for the reconstruction. To utilize the Fourier method locally based on the local non-periodic data, the proposed method is based on the Fourier continuation method. This work is an extension of our previous 1D Fourier domain decomposition method to 2D Fourier data. The proposed method first divides the MRI image in the spatial domain into many subdomains and applies the Fourier continuation method for the smooth periodic extension of the subdomain of interest. Then the proposed method reconstructs the local image based on L2 minimization regularized by the L1 norm of edge sparsity to sharpen the image near edges. Our numerical results suggest that the proposed method should be utilized in dimension-by-dimension manner instead of in a global manner for both the quality of the reconstruction and computational efficiency. The numerical results show that the proposed method is effective when the local reconstruction is sought and that the solution is free of Gibbs oscillations.

neuroscience↗

bioRxiv2018

PBMC Fixation and Processing for Chromium Single-Cell RNA Sequencing

BackgroundInterest in single-cell transcriptomic analysis is growing rapidly, especially for profiling rare or heterogeneous populations of cells. In almost all reported works investigators have used live cells, which introduces cell stress during preparation and hinders complex study designs. Recent studies have indicated that cells fixed by denaturing fixative can be used in single-cell sequencing, however they did not usually work with most types of primary cells including immune cells.\n\nMethodsThe methanol-fixation and new processing method was introduced to preserve human peripheral blood mononuclear cells (PBMCs) for single-cell RNA sequencing (scRNA-Seq) analysis on 10X Chromium platform.\n\nResultsWhen methanol fixation protocol was broken up into three steps: fixation, storage and rehydration, we found that PBMC RNA was degraded during rehydration with PBS, not at cell fixation and up to three-month storage steps. Resuspension but not rehydration in 3X saline sodium citrate (SSC) buffer instead of PBS preserved PBMC RNA integrity and prevented RNA leakage. Diluted SSC buffer did not interfere with full-length cDNA synthesis. The methanol-fixed PBMCs resuspended in 3X SSC were successfully implemented into 10X Chromium standard scRNA-seq workflows with no elevated low quality cells and cell doublets. The fixation process did not alter the single-cell transcriptional profiles and gene expression levels. Major subpopulations classified by marker genes could be identified in fixed PBMCs at a similar proportion as in live PBMCs. This new fixation processing protocol also worked in several other fixed primary cell types and cell lines as in live ones.\n\nConclusionsWe expect that the methanol-based cell fixation procedure presented here will allow better and more effective batching schemes for a complex single cell experimental design with primary cells or tissues.

genomics↗

bioRxiv2018

Programmable single and multiplex base-editing in Bombyx mori using RNA-guided cytidine deaminases

Standard genome editing tools (ZFN, TALEN and CRISPR/Cas9) edited genome depending on DNA double strand breaks (DSBs). A series of new CRISPR tools that convert cytidine to thymine (C to T) without the requirement for DNA double-strand breaks were developed recently, which have changed this status and have been quickly applied in a variety of organisms. Here, we demonstrate that CRISPR/Cas9-dependent base editor (BE3) converts C to T with a high frequency in the invertebrate Bombyx mori silkworm. Using BE3 as a knock-out tool, we inactivated exogenous and endogenous genes through base-editing-induced nonsense mutations with an efficiency of up to 66.2%. Furthermore, genome-scale analysis showed that 96.5% of B. mori genes have one or more targetable sites being knocked out by BE3 with a median of 11 sites per gene. The editing window of BE3 reached up to 13 bases (from C1 to C13 in the range of gRNA) in B. mori. Notably, up to 14 bases were substituted simultaneously in a single DNA molecule, with a low indel frequency of 0.6%, when 32 gRNAs were co-transfected. Collectively, our data show for the first time that RNA-guided cytidine deaminases are capable of programmable single and multiplex base-editing in an invertebrate model.

genetics↗