RESUMO
Tagmentation combines DNA fragmentation and sequencing adapter addition by leveraging the transposition activity of the bacterial cut-and-paste Tn5 transposase, to enable efficient sequencing library preparation. Here we present an open-source protocol for the generation of multi-purpose hyperactive Tn5 transposase, including its benchmarking in CUT&Tag, bulk and single-cell ATAC-seq. The OpenTn5 protocol yields multi-milligram quantities of pG-Tn5E54K, L372P protein per liter of E. coli culture, sufficient for thousands of tagmentation reactions and the enzyme retains activity in storage for more than a year.
RESUMO
Cis-regulatory elements control gene expression and are dynamic in their structure, reflecting changes to the composition of diverse effector proteins over time1-3. Here we sought to connect the structural changes at cis-regulatory elements to alterations in cellular fate and function. To do this we developed PRINT, a computational method that uses deep learning to correct sequence bias in chromatin accessibility data and identifies multi-scale footprints of DNA-protein interactions. We find that multi-scale footprints enable more accurate inference of TF and nucleosome binding. Using PRINT with single-cell multi-omics, we discover wide-spread changes to the structure and function of candidate cis-regulatory elements (cCREs) across hematopoiesis, wherein nucleosomes slide, expose DNA for TF binding, and promote gene expression. Activity segmentation using the co-variance across cell states identifies "sub-cCREs" as modular cCRE subunits of regulatory DNA. We apply this single-cell and PRINT approach to characterize the age-associated alterations to cCREs within hematopoietic stem cells (HSCs). Remarkably, we find a spectrum of aging alterations among HSCs corresponding to a global gain of sub-cCRE activity while preserving cCRE accessibility. Collectively, we reveal the functional importance of cCRE structure across cell states, highlighting changes to gene regulation at single-cell and single-base-pair resolution.
RESUMO
The ruthenium phosphite hydride complexes H(2)Ru(P(OR)(3))(4) (R = Me (1), Et (2), (i)Pr (3)) were used as CVD precursors for the deposition of films of amorphous ruthenium-phosphorus alloys. The as-deposited films were X-ray amorphous and XPS analysis revealed that they were predominantly comprised of Ru and P in zero oxidation states. XPS analysis also showed the presence of small amounts of oxidized ruthenium and phosphorus. The composition of the films was found to depend on ligand chemistry as well as the deposition conditions. The use of H(2) as the carrier gas had the effect of increasing the relative concentrations of P and O for all films. Annealing films to 700 °C under vacuum produced films of polycrystalline hcp Ru while a flowing stream of H(2) resulted in polycrystalline hcp RuP.
RESUMO
New dinuclear rhodium(II) pyrazolate (Pz) complexes of formula Rh(2)(3-R,5-R'Pz)(4)·2L (R = R' = CF(3), L = H(2)O (1), CH(3)CN (2)) and Rh(2)(3-R,5-R'Pz)(4) (R = R' = (t)Bu (3); R = CF(3), R' = (t)Bu (4)) have been synthesized from the interaction of the lithium salt of the corresponding pyrazole with Rh(2)(OAc)(4) in diethyl ether. The complexes were characterized by X-ray crystallography and spectroscopic methods. They were further evaluated as precursors for the chemical vapor deposition (CVD) of Rh thin films using H(2) as the carrier gas. The resulting films were characterized by scanning electron microscopy (SEM), X-ray diffraction (XRD) and X-ray photoelectron spectroscopy (XPS).
RESUMO
Reaction of 3,5-(CF(3))(2)PzLi with [Rh(µ-Cl)(η(2)-C(2)H(4))(2)](2) or [Rh(µ-Cl)(PMe(3))(2)](2) in Et(2)O gave the dinuclear complexes [Rh(η(2)-C(2)H(4))(2)(µ-3,5-(CF(3))(2)-Pz)](2) (1) and [Rh(2)(µ-Cl)(µ-3,5-(CF(3))(2)-Pz) (PMe(3))(4)] (2) respectively (3,5-(CF(3))(2)Pz = bis-trifluoromethyl pyrazolate). Reaction of PMe(3) with [Rh(COD)(µ-3,5-(CF(3))(2)-Pz)](2) in toluene gave [Rh(3,5-(CF(3))(2)-Pz)(PMe(3))(3)] (3). Reaction of 1 and 3 in toluene (1 : 4) gave moderate yields of the dinuclear complex [Rh(PMe(3))(2)(µ-3,5-(CF(3))(2)-Pz)](2) (4). Reaction of 3,5-(CF(3))(2)PzLi with [Rh(PMe(3))(4)]Cl in Et(2)O gave the ionic complex [Rh(PMe(3))(4)][3,5-(CF(3))(2)-Pz] (5). Two of the complexes, 1 and 3, were studied for use as CVD precursors. Polycrystalline thin films of rhodium (fcc-Rh) and metastable-amorphous films of rhodium phosphide (Rh(2)P) were grown from 1 and 3 respectively at 170 and 130 °C, 0.3 mmHg in a hot wall reactor using Ar as the carrier gas (5 cc min(-1)). Thin films of amorphous rhodium and rhodium phosphide (Rh(2)P) were grown from 1 and 3 at 170 and 130 °C respectively at 0.3 mmHg in a hot wall reactor using H(2) as the carrier gas (7 cc min(-1)).