Single-Cell RNA Sequencing Analysis: A Step-by-Step Overview

Thanks to innovative sample-preparation and sequencing technologies, gene expression in individual cells can now be measured for thousands of cells in a single experiment. Since its introduction, single-cell RNA sequencing (scRNA-seq) approaches have revolutionized the genomics field as they created unprecedented opportunities for resolving cell heterogeneity by exploring gene expression profiles at a single-cell resolution. However, the rapidly evolving field of scRNA-seq invoked the emergence of various analytics approaches aimed to maximize the full potential of this novel strategy. Unlike population-based RNA sequencing approaches, scRNA seq necessitates comprehensive computational tools to address high data complexity and keep up with the emerging single-cell associated challenges. Despite the vast number of analytical methods, a universal standardization is lacking. While this reflects the fields’ immaturity, it may also encumber a newcomer to blend in. In this review, we aim to bridge over the abovementioned hurdle and propose four ready-to-use pipelines for scRNA-seq analysis easily accessible by a newcomer, that could fit various biological data types. Here we provide an overview of the currently available single-cell technologies for cell isolation and library preparation and a step by step guide that covers the entire canonical analytic workflow to analyse scRNA-seq data including read mapping, quality controls, gene expression quantification, normalization, feature selection, dimensionality reduction, and cell clustering useful for trajectory inference and differential expression. Such workflow guidelines will escort novices as well as expert users in the analysis of complex scRNA-seq datasets, thus further expanding the research potential of single-cell approaches in basic science, and envisaging its future implementation as best practice in the field

Post-transcriptional gene silencing triggered by sense transgenes involves uncapped antisense RNA and differs from silencing intentionally triggered by antisense transgenes

Although post-transcriptional gene silencing (PTGS) has been studied for more than a decade, there is still a gap in our understanding of how de novo silencing is initiated against genetic elements that are not supposed to produce double-stranded (ds)RNA. Given the pervasive transcription occurring throughout eukaryote genomes, we tested the hypothesis that unintended transcription could produce antisense (as)RNA molecules that participate to the initiation of PTGS triggered by sense transgenes (S-PTGS). Our results reveal a higher level of asRNA in Arabidopsis thaliana lines that spontaneously trigger S-PTGS than in lines that do not. However, PTGS triggered by antisense transgenes (AS-PTGS) differs from S-PTGS. In particular, a hypomorphic ago1 mutation that suppresses S-PTGS prevents the degradation of asRNA but not sense RNA during AS-PTGS, suggesting a different treatment of coding and non-coding RNA by AGO1, likely because of AGO1 association to polysomes. Moreover, the intended asRNA produced during AS-PTGS is capped whereas the asRNA produced during S-PTGS derives from 3' maturation of a read-through transcript and is uncapped. Thus, we propose that uncapped asRNA corresponds to the aberrant RNA molecule that is converted to dsRNA by RNA-DEPENDENT RNA POLYMERASE 6 in siRNA-bodies to initiate S-PTGS, whereas capped asRNA must anneal with sense RNA to produce dsRNA that initiate AS-PTGS

Non variability of intervening absorbers observed in the UVES spectra of the "naked-eye" GRB080319

The aim of this paper is to investigate the properties of the intervening absorbers lying along the line of sight of Gamma-Ray Burst (GRB) 080319B through the analysis of its optical absorption features. To this purpose, we analyze a multi-epoch, high resolution spectroscopic observations (R=40000, corresponding to 7.5 km/s) of the optical afterglow of GRB080319B (z=0.937), taken with UVES at the VLT. Thanks to the rapid response mode (RRM), we observed the afterglow just 8m:30s after the GRB onset when the magnitude was R ~ 12. This allowed us to obtain the best signal-to-noise, high resolution spectrum of a GRB afterglow ever (S/N per resolution element ~ 50). Two further RRM and target of opportunity observations were obtained starting 1.0 and 2.4 hours after the event, respectively. Four MgII absorption systems lying along the line of sight to the afterglow have been detected in the redshift range 0.5 < z < 0.8, most of them showing a complex structure featuring several components. Absorptions due to FeII, MgI and MnII are also present; they appear in four, two and one intervening absorbers, respectively. One out of four systems show a MgII2796 rest frame equivalent width larger than 1A. This confirms the excess of strong MgII absorbers compared to quasars, with dn/dz = 0.9, ~ 4 times larger than the one observed along quasar lines of sight. In addition, the analysis of multi-epoch, high-resolution spectra allowed us to exclude a significant variability in the column density of the single components of each absorber. Combining this result with estimates of the size of the emitting region, we can reject the hypothesis that the difference between GRB and QSO MgII absorbers is due to a different size of the emitting regions.Comment: 10 pages, 15 ps figures, submitted to MNRA

The Blue Straggler population in the globular cluster M53 (NGC5024): a combined HST, LBT, CFHT study

We used a proper combination of multiband high-resolution and wide field multi-wavelength observations collected at three different telescopes (HST, LBT and CFHT) to probe Blue Straggler Star (BSS) populations in the globular cluster M53. Almost 200 BSS have been identified over the entire cluster extension. The radial distribution of these stars has been found to be bimodal (similarly to that of several other clusters) with a prominent dip at ~60'' (~2 r_c) from the cluster center. This value turns out to be a factor of two smaller than the radius of avoidance (r_avoid, the radius within which all the stars of ~1.2 M_sun have sunk to the core because of dynamical friction effects in an Hubble time). While in most of the clusters with a bimodal BSS radial distribution, r_avoid has been found to be located in the region of the observed minimum, this is the second case (after NGC6388) where this discrepancy is noted. This evidence suggests that in a few clusters the dynamical friction seems to be somehow less efficient than expected. We have also used this data base to construct the radial star density profile of the cluster: this is the most extended and accurate radial profile ever published for this cluster, including detailed star counts in the very inner region. The star density profile is reproduced by a standard King Model with an extended core (~25'') and a modest value of the concentration parameter (c=1.58). A deviation from the model is noted in the most external region of the cluster (at r>6.5' from the center). This feature needs to be further investigated in order to address the possible presence of a tidal tail in this cluster.Comment: 25 pages, 9 figures, accepted for publication on Ap

Measuring the Halo Mass of z=3 Damped Ly-alpha Absorbers from the Absorber-Galaxy Cross-correlation

[Abridged] We test the reliability of a method to measure the mean halo mass of Damped Ly-alpha absorbers (DLAs). The method is based on measuring the ratio of the cross-correlation between DLAs and galaxies to the auto-correlation of the galaxies themselves ($w_{\rm dg}/w_{\rm gg}$), which is (in linear theory) the ratio of their bias factor. This is shown to be true irrespective of the galaxy redshift distribution, provided that one uses the same galaxies for the two correlation functions. The method is applicable to all redshifts. Here, we focus on z=3 DLAs and we demonstrate that the method robustly constrains the mean DLA halo mass using smoothed particle hydrodynamics (SPH) cosmological simulations. If we use the bias formalism of Mo & White with the DLA and galaxy mass distributions of these simulations, we predict a bias ratio of 0.771. Direct measurement from the simulations of $w_{\rm dg}/w_{\rm gg}$ st yields a ratio of 0.73+/-0.08, in excellent agreement with that prediction. Equivalently, inverting the measured correlation ratio to infer a mean DLA halo mass yields (log. averaging, in solar units) =11.13+/-013, in excellent agreement with the true value in the simulations: 11.16. The cross- correlation method thus appears to yield a robust estimate of the average host halo mass even though the DLAs and the galaxies occupy a broad mass spectrum of halos, and massive halos contain multiple galaxies with DLAs. We show that the inferred mean DLA halo mass is independent of the galaxy sub-sample used, i.e. the cross-correlation technique is also reliable. Our results imply that the cross-correlation length between DLAs and LBGs is predicted to be, at most, 2.85 Mpc. Future observations will soon distinguish models in which DLAs are in low mass halos from those in which DLAs are in massive halos.Comment: 15 pages, 7 figures, to be published in ApJ 2005 July 20th (Full resolution of Fig.2 at http://www.mpe.mpg.de/~nbouche/papers/Xcorr/f2-orig.eps); minor changes to match the published tex

Mixing in a swarm of bubbles rising in a confined cell measured by mean of PLIF with two different dyes

The present contribution reports an experimental study of the mixing of a passive scalar of very low diffusivity in a homogeneous swarm of inertial bubbles rising in a thin gap. A patch of fluorescent dye is injected within the swarm, and we observe the evolution of its mass in a given region of observation. We analyse the effect of the liquid agitation on the mixing mechanisms varying the gas volume fraction from 1.3 to 7.5 %, while the Reynolds number of the bubbles, Re = 450, their Weber number, We = 0.7, and the gapto-bubble diameter ratio, w/d = 0.25, are kept approximately constant. Here, the in-plane local mass of dye is measured by using a two-dyes planar laser-induced fluorescence (PLIF) technique that has been adapted to fix the problem of multiple light reflections at the bubble interfaces. Indeed, they induce both temporal and spatial variations of the captured light intensity that are superimposed to the effective fluorescence signal and prevent from using a standard PLIF technique. The analysis of the instantaneous concentration fields reveals the dominant role of the bubble wakes in the scalar transport. It is shown that mixing in this planar confined geometry is very efficient and enhanced by the increasing gas volume fraction. The present study also highlights that the mixing is not governed by a Fickian law of diffusion

The Role of Sub-damped Lyman-alpha Absorbers in the Cosmic Evolution of Metals

Observations of low mean metallicity of damped Lyman-alpha (DLA) quasar absorbers at all redshifts studied appear to contradict the predictions for the global mean interstellar metallicity in galaxies from cosmic chemical evolution models. On the other hand, a number of metal-rich sub-DLA systems have been identified recently, and the fraction of metal-rich sub-DLAs appears to be considerably larger than that of metal-rich DLAs, especially at z < 1.5. In view of this, here we investigate the evolution of metallicity in sub-DLAs. We find that the mean Zn metallicity of the observed sub-DLAs may be higher than that of the observed DLAs, especially at low redshifts, reaching a near-solar level at z <~ 1. This trend does not appear to be an artifact of sample selection, the use of Zn, the use of N_{HI}-weighting, or observational sensitivity. While a bias against very low metallicity could be present in the sub-DLA sample in some situations, this cannot explain the difference between the DLA and sub-DLA metallicities at low z. The primary reason for the difference between the DLAs and sub-DLAs appears to be the dearth of metal-rich DLAs. We estimate the sub-DLA contribution to the total metal budget using measures of their metallicity and comoving gas density. These calculations suggest that at z <~ 1, the contribution of sub-DLAs to the total metal budget may be several times that of DLAs. At higher redshifts also, there are indications that the sub-DLAs may contribute significantly to the cosmic metal budget.Comment: 9 pages, 2 figures, Accepted for Publication in the Astrophysical Journa

Non-Equilibrium Quantum Fields in the Large N Expansion

An effective action technique for the time evolution of a closed system consisting of one or more mean fields interacting with their quantum fluctuations is presented. By marrying large $N$ expansion methods to the Schwinger-Keldysh closed time path (CTP) formulation of the quantum effective action, causality of the resulting equations of motion is ensured and a systematic, energy conserving and gauge invariant expansion about the quasi-classical mean field(s) in powers of $1/N$ developed. The general method is exposed in two specific examples, $O(N)$ symmetric scalar \l\F^4 theory and Quantum Electrodynamics (QED) with $N$ fermion fields. The \l\F^4 case is well suited to the numerical study of the real time dynamics of phase transitions characterized by a scalar order parameter. In QED the technique may be used to study the quantum non-equilibrium effects of pair creation in strong electric fields and the scattering and transport processes in a relativistic $e^+e^-$ plasma. A simple renormalization scheme that makes practical the numerical solution of the equations of motion of these and other field theories is described.Comment: 43 pages, LA-UR-94-783 (PRD, in press), uuencoded PostScrip

RDR2 Partially Antagonizes the Production of RDR6-Dependent siRNA in Sense Transgene-Mediated PTGS

Background: RNA-DEPENDENT RNA POLYMERASE6 (RDR6) and SUPPRESSOR of GENE SILENCING 3 (SGS3) are required for DNA methylation and post-transcriptional gene silencing (PTGS) mediated by 21-nt siRNAs produced by sense transgenes (S-PTGS). In contrast, RDR2, but not RDR6, is required for DNA methylation and TGS mediated by 24-nt siRNAs, and for cellto-cell spreading of IR-PTGS mediated by 21-nt siRNAs produced by inverted repeat transgenes under the control of a phloem-specific promoter. Principal Findings: In this study, we examined the role of RDR2 and RDR6 in S-PTGS. Unlike RDR6, RDR2 is not required for DNA methylation of transgenes subjected to S-PTGS. RDR6 is essential for the production of siRNAs by transgenes subjected to S-PTGS, but RDR2 also contributes to the production of transgene siRNAs when RDR6 is present because rdr2 mutations reduce transgene siRNA accumulation. However, the siRNAs produced via RDR2 likely are counteractive in wildtype plants because impairement of RDR2 increases S-PTGS efficiency at a transgenic locus that triggers limited silencing, and accelerates S-PTGS at a transgenic locus that triggers efficient silencing. Conclusions/Significance: These results suggest that RDR2 and RDR6 compete for RNA substrates produced by transgenes subjected to S-PTGS. RDR2 partially antagonizes RDR6 because RDR2 action likely results in the production of counteractiv