1.
PBMC fixation and processing for Chromium single-cell RNA sequencing.
Chen, J, Cheung, F, Shi, R, Zhou, H, Lu, W, ,
Journal of translational medicine. 2018;(1):198
Abstract
BACKGROUND Interest 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. METHODS The 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 10× Chromium platform. RESULTS When 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 3-month storage steps. Resuspension but not rehydration in 3× 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 3× SSC were successfully implemented into 10× 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. CONCLUSIONS We 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.
2.
Geophysical monitoring of hydrological and biogeochemical transformations associated with Cr(VI) bioremediation.
Hubbard, SS, Williams, K, Conrad, ME, Faybishenko, B, Peterson, J, Chen, J, Long, P, Hazent, T
Environmental science & technology. 2008;(10):3757-65
Abstract
Understanding how hydrological and biogeochemical properties change over space and time in response to remedial treatments is hindered by our ability to monitor these processes with sufficient resolution and over field relevant scales. Here, we explored the use of geophysical approaches for monitoring the spatiotemporal distribution of hydrological and biogeochemical transformations associated with a Cr(VI) bioremediation experiment performed at Hanford, WA. We first integrated hydrological wellbore and geophysical tomographic data sets to estimate hydrological zonation at the study site. Using results from laboratory biogeophysical experiments and constraints provided by field geochemical data sets, we then interpreted time-lapse seismic and radar tomographic data sets, collected during thirteen acquisition campaigns over a three year experimental period, in terms of hydrological and biogeochemical transformations. The geophysical monitoring data sets were used to infer: the spatial distribution of injected electron donor; the evolution of gas bubbles; variations in total dissolved solids (nitrate and sulfate) as a function of pumping activity; the formation of precipitates and dissolution of calcites; and concomitant changes in porosity. Although qualitative in nature, the integrated interpretation illustrates how geophysical techniques have the potential to provide a wealth of information about coupled hydrobiogeochemical responses to remedial treatments in high spatial resolution and in a minimally invasive manner. Particularly novel aspects of our study include the use of multiple lines of evidence to constrain the interpretation of a long-term, field-scale geophysical monitoring data set and the interpretation of the transformations as a function of hydrological heterogeneity and pumping activity.