The striatum contributes to many cognitive processes and disorders, but its cell types are incompletely characterized. We show that microfluidic and FACS-based single-cell RNA sequencing of mouse striatum provides a well-resolved classification of striatal cell type diversity. Transcriptome analysis revealed 10 differentiated distinct cell types, including neurons, astrocytes, oligodendrocytes, ependymal, immune, and vascular cells, and enabled the discovery of numerous marker genes. Furthermore, we identified two discrete subtypes of medium spiny neurons (MSN) which have specific markers and which overexpress genes linked to cognitive disorders and addiction. We also describe continuous cellular identities, which increase heterogeneity within discrete cell types. Finally, we identified cell type specific transcription and splicing factors that shape cellular identities by regulating splicing and expression patterns. Our findings suggest that functional diversity within a complex tissue arises from a small number of discrete cell types, which can exist in a continuous spectrum of functional states.

Microinfarctions are present in the aged and injured human brain. Their clinical significance is controversial, with postulated sequelae ranging from cognitive sparing to vascular dementia. To address the consequences of microinfarcts, we used controlled optical methods to create occlusions of individual penetrating arterioles or venules within rat cortex. Single microinfarcts, targeted to encompass all or part of a cortical column, impaired performance in a macrovibrissa-based behavioral task. Further, multiple targeted vessels caused tissue damage that coalesced across cortex, even though the intervening penetrating vessels were acutely patent. Post-occlusion administration of Memantine, a glutamate receptor antagonist that reduces cognitive decline in Alzheimer’s disease, ameliorated tissue damage and perceptual deficits. Collectively, these data imply that microinfarcts are likely contributors to cognitive decline. Strategies that have received limited success in the treatment of ischemic injury, which include therapeutics against excitotoxicity, may be successful against the progressive nature of vascular dementia.

Arteries and veins are specified by antagonistic transcriptional programs. However, during development and regeneration, new arteries can arise from pre-existing veins through a poorly understood process of cell fate conversion. Here, using single-cell RNA sequencing and mouse genetics, we show that vein cells of the developing heart undergo an early cell fate switch to create a pre-artery population that subsequently builds coronary arteries. Vein cells underwent a gradual and simultaneous switch from venous to arterial fate before a subset of cells crossed a transcriptional threshold into the pre-artery state. Before the onset of coronary blood flow, pre-artery cells appeared in the immature vessel plexus, expressed mature artery markers, and decreased cell cycling. The vein-specifying transcription factor COUP-TF2 (also known as NR2F2) prevented plexus cells from overcoming the pre-artery threshold by inducing cell cycle genes. Thus, vein-derived coronary arteries are built by pre-artery cells that can differentiate independently of blood flow upon the release of inhibition mediated by COUP-TF2 and cell cycle factors.

There is a need in molecular biology and biomedical research for open-ended, hypothesis-generating research, in order to discover previously unknown molecular mechanisms. Genetic screening provides a powerful approach for identifying genes, pathways and mechanisms involved in a given phenotype or biological process. This is illustrated by the many successes of forward genetics in cell lines 1 and in model organisms such as flies 2,3 , worms 4 , yeast 5 , plants 6 and fish 7 , and pioneering work in RNA interference (RNAi) screens 8,9 .CRISPR screens exploit the efficiency and flexibility of CRISPR-Cas genome editing 10 . They have become a popular and productive tool for biological discovery in a broad range of applications 11,12 . In a typical pooled CRISPR screen (FIg. 1), a CRISPR guide RNA (gRNA) library is introduced in bulk into cells, such that individual cells receive different gRNAs and are perturbed according to the gRNA received by the cell. These gRNAs are usually delivered by lentiviral transduction and are integrated into the DNA of the target cells, making it possible to efficiently determine the induced perturbations based on the gRNA sequence.

Illumina-based next generation sequencing (NGS) has accelerated biomedical discovery through its ability to generate thousands of gigabases of sequencing output per run at a fraction of the time and cost of conventional technologies. The process typically involves four basic steps: library preparation, cluster generation, sequencing, and data analysis. In 2015, a new chemistry of cluster generation was introduced in the newer Illumina machines (HiSeq 3000/4000/X Ten) called exclusion amplification (ExAmp), which was a fundamental shift from the earlier method of random cluster generation by bridge amplification on a non-patterned flow cell. The ExAmp chemistry, in conjunction with patterned flow cells containing nanowells at fixed locations, increases cluster density on the flow cell, thereby reducing the cost per run. It also increases sequence read quality, especially for longer read lengths (up to 150 base pairs). This advance has been widely adopted for genome sequencing because greater sequencing depth can be achieved for lower cost without compromising the quality of longer reads. We show that this promising chemistry is problematic, however, when multiplexing samples. We discovered that up to 5-10% of sequencing reads (or signals) are incorrectly assigned from a given sample to other samples in a multiplexed pool. We provide evidence that this "spreading-of-signals" arises from low levels of free index primers present in the pool. These index primers can prime pooled library fragments at random via complementary 3' ends, and get extended by DNA polymerase, creating a new library molecule with a new index before binding to the patterned flow cell to generate a cluster for sequencing. This causes the resulting read from that cluster to be assigned to a different sample, causing the spread of signals within multiplexed samples. We show that low levels of free index primers persist after the most common library purification procedure recommended by Illumina, and that the amount of signal spreading among samples is proportional to the level of free index primer present in the library pool. This artifact causes homogenization and misclassification of cells in single cell RNA-seq experiments. Therefore, all data generated in this way must now be carefully re-examined to ensure that "spreading-ofsignals" has not compromised data analysis and conclusions. Re-sequencing samples using an older technology that uses conventional bridge amplification for cluster generation, or improved library cleanup strategies to remove free index primers, can minimize or eliminate this signal spreading artifact.