Thanks to recent technological advances in X-ray and micro-electron diffraction and solid-state NMR, structural information can be obtained by using much smaller crystals. Thus, microcrystals have become a valuable commodity rather than a mere stepping stone toward obtaining macroscopic crystals. Microcrystals are particularly useful for structure determination using serial data collection approaches at synchrotrons and X-ray free-electron lasers. The latter's enormous peak brilliance and short X-ray pulse duration mean that structural information can be obtained before the effects of radiation damage are seen; these properties also facilitate time-resolved crystallography. To establish defined reaction initiation conditions, microcrystals with a desired and narrow size distribution are critical. Here, we describe milling and seeding techniques as well as filtration approaches for the reproducible and size-adjustable preparation of homogeneous nano- and microcrystals. Nanocrystals and crystal seeds can be obtained by milling using zirconium beads and the BeadBug homogenizer; fragmentation of large crystals yields micro- or nanocrystals by flowing crystals through stainless steel filters by using an HPLC pump. The approaches can be scaled to generate micro- to milliliter quantities of microcrystals, starting from macroscopic crystals. The procedure typically takes 3-5 d, including the time required to grow the microcrystals.

Single-cell RNA-sequencing (scRNA-seq) allows the characterization of cellular composition and interactions in complex tissues. An essential prerequisite for scRNA-seq is the preparation of high-quality single-cell suspensions. So far, no protocols have been described for preparing such suspensions from the placenta, an essential organ for fetal development and a site of maternal-fetal immune interaction. Here we describe a protocol for the preparation of high-quality single-cell suspensions from human placental tissues-namely, the basal plate, placental villi and chorioamniotic membranes. The protocol outlines the collection of tissues from the placenta, tailored dissociation procedures for each tissue, and the cryopreservation of single-cell suspensions for multiplex sequencing library preparation. The protocol can be performed by a qualified investigator with basic working knowledge of placental structure. Moreover, the single-cell suspensions generated by using this protocol are compatible with droplet-based scRNA-seq technology, such as the 10x Genomics Chromium system. This protocol reliably produces single-cell suspensions from the placental tissues with high yield and viability for scRNA-seq. This protocol takes ~6 h to complete from tissue collection to cryopreservation of single-cell suspensions, and an additional 2 h for thawing of cryopreserved single cells.

The chromosome conformation capture method and its derivatives, such as circularized chromosome conformation capture, carbon copy chromosome conformation capture, high-throughput chromosome conformation capture and capture high-throughput chromosome conformation capture, have pioneered our understanding of the principles of chromosome folding in the nucleus. These technical advances, however, cannot precisely quantitate interaction frequency in very small input samples. Here we describe a protocol for the Nodewalk assay, which is based on converting chromosome conformation capture DNA samples to RNA and subsequently to cDNA using strategically placed primers. This pipeline enables the quantitative analyses of chromatin fiber interactions without compromising its sensitivity down to <300 cells, making it suitable for MiSeq analyses of higher-order chromatin structures in biopsies, circulating tumor cells and transitional cell states, for example. Importantly, the quality of the Nodewalk sample can be assessed before sequencing to avoid unnecessary costs. Moreover, it enables analyses from hundreds of different restriction enzyme fragment viewpoints within the same initial small input sample to uncover complex, genome-wide networks. Following optimization of the different steps, the entire protocol can be completed within 2 weeks.

Prime editors (PEs), which can install desired base edits without donor DNA or double-strand breaks, have been used in plants and can, in principle, accelerate crop improvement and breeding. However, their editing efficiency in plants is generally low. Optimizing the prime editing guide RNA (pegRNA) by designing the sequence on the basis of melting temperature, using dual-pegRNAs and engineering PEs have all been shown to enhance PE efficiency. In addition, an automated pegRNA design platform, PlantPegDesigner, has been developed on the basis of rice prime editing experimental data. In this protocol, we present detailed protocols for designing and optimizing pegRNAs using PlantPegDesigner, constructing engineered plant PE vectors with enhanced editing efficiency for prime editing, evaluating prime editing efficiencies using a reporter system and comparing the effectiveness and byproducts of PEs by deep amplicon sequencing. Using this protocol, researchers can construct optimized pegRNAs for prime editing in 4-7 d and obtain prime-edited rice or wheat plants within 3 months.