Protocol for mapping RNA G-quadruplex for chromatin-bound RNA using d-rG4-seq.

Here, we present a protocol for using d-rG4-seq, a technique for mapping RNA G-quadruplex (rG4) for chromatin-bound RNA. We describe steps for identifying in vivo rG4 structures based on differential sensitivity of rG4 to dimethyl sulfate (DMS) modification, folding in the presence of monovalent cations, K+ versus Li+, and reverse transcriptase (RT) readthrough when folded. We then detail procedures for isolating RNA from the chromatin-bound fractions to enrich for epigenetic regulators and comparing in vitro versus in vivo profiles.

Denaturing cross-linking immunoprecipitation to identify footprints for RNA-binding proteins.

The isolation of protein-RNA complexes in the "denaturing cross-linked RNA immunoprecipitation" (dCLIP) protocol is based on biotin-tagging proteins of interest, UV cross-linking RNA to protein in vivo, RNase protection assay, and isolating RNA-protein complexes under denaturing conditions over a streptavidin column. Insofar as conventional antibody-based CLIP assays have been challenging to apply to Polycomb complexes, dCLIP has been applied successfully and yields small RNA footprints from which de novo motif analysis can be performed to identify RNA binding motifs. For complete details on the use and execution of this protocol, please refer to Rosenberg et al.

Epidermal stem cells arise from the hair follicle after wounding.

During normal development, the epidermis and hair follicle are distinct lineage compartments maintained by independent stem cell populations. Both epidermal and follicular keratinocytes are recruited to participate in epidermal repair in response to injury. However, it is generally thought that follicular cells contribute to the wound epidermis only transiently and are ultimately replaced by the progeny of stem cells derived from the original epidermal compartment prior to wounding.

Distinct stem cell populations regenerate the follicle and interfollicular epidermis.

The regeneration of the skin and its appendages is thought to occur by the regulated activation of a dedicated stem cell population. A population of cells in the bulge region of the hair follicle has been identified as the putative stem cell of both the follicle and the interfollicular epidermis. While this assertion is supported by a variety of surrogate assays, there has been no direct confirmation of the normal contribution of these cells to the regeneration of structures other than the cycling portion of the hair follicle.

Temporal analysis of the early BMP functions identifies distinct anti-organizer and mesoderm patterning phases.

BMP signaling performs multiple important roles during early embryogenesis. Signaling through the BMP pathway is mediated by different BMP ligands expressed in partially overlapping temporal and spatial patterns. Assignment of different BMP-dependent activities to the individual ligands has relied on the patterns of expression of the various BMP genes.

The competence of marginal zone cells to become Spemann's organizer is controlled by Xcad2.

The organizer in vertebrate embryos is responsible for the formation of the primary body axis. In amphibian embryos, the organizer forms in the dorsal marginal zone (prospective dorsal mesoderm) at a location determined by the point of sperm entry. Using inducible versions of axis-inducing proteins, it has been shown that, irrespective of the mode of secondary axis induction, organizer formation in the ventral marginal zone is temporally restricted from the midblastula transition to the onset of gastrulation.