Dataset Information

BRD4 regulates the osteogenic differentiation of MSCs

ABSTRACT: MSCs were lysed in cell lysis buffer for Western blotting and IP (Beyotime, Cat. No. P0013). A Dynabeads™ protein G immunoprecipitation kit (Invitrogen, Cat. No. 10007D) was used for Co-IP according to the manufacturer’s instructions. Dynabeads were resuspended and placed on a magnet to remove the supernatant then incubated with Ab Binding & Washing Buffer containing an anti-BRD4 antibody for 10 min at room temperature with rotation. The supernatant was removed by placing tubes on a magnet, and the Dynabeads-antibody complex was washed with Ab Binding & Washing Buffer. MSC lysates were added to the Dynabeads-antibody mixture and incubated for 10 min at room temperature with rotation. The supernatant was removed, the Dynabeads-antibody complex mixture was washed with washing buffer, and the proteins were separated using a standard SDS–PAGE system.

ORGANISM(S): Homo Sapiens

SUBMITTER: Huiyong Shen

PROVIDER: PXD034615 | iProX | Thu Jun 16 00:00:00 BST 2022

REPOSITORIES: iProX

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Publications

Super enhancers targeting ZBTB16 in osteogenesis protect against osteoporosis.

Yu Wenhui W Xie Zhongyu Z Li Jinteng J Lin Jiajie J Su Zepeng Z Che Yunshu Y Ye Feng F Zhang Zhaoqiang Z Xu Peitao P Zeng Yipeng Y Xu Xiaojun X Li Zhikun Z Feng Pei P Mi Rujia R Wu Yanfeng Y Shen Huiyong H

Bone research 20230607 1

As the major cell precursors in osteogenesis, mesenchymal stem cells (MSCs) are indispensable for bone homeostasis and development. However, the primary mechanisms regulating osteogenic differentiation are controversial. Composed of multiple constituent enhancers, super enhancers (SEs) are powerful cis-regulatory elements that identify genes that ensure sequential differentiation. The present study demonstrated that SEs were indispensable for MSC osteogenesis and involved in osteoporosis develop ...[more]

PMID: 37280207

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Project description:The modified iCLIP protocol is based on the previously described protocol (Huppertz et al., 2014) with modifications that enable the definition of readthrough cDNAs. HeLa cells were crosslinked with 0.15mJ/cm2 254nm UV light. Protein G Dynabeads were used for immunoprecipitations (IP). For each IP, 100 l of beads were washed in iCLIP lysis buffer (50mM Tris-HCL pH 7.4, 100mM NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate), and incubated with the anti-eIF4A3 antibody or polyclonal mAb BB7 serum anti-PTBP1. To prepare the cell lysate, the cells were lysed with 1 mL iCLIP lysis buffer (final concentration 2mg/mL), sonicated (Bioruptor, 5x5 sec on/off), incubated with RNase I (1-2 x10-3 U/mL for eIF4A3) at 37C for 3 min, and centrifuged. After the pellet had dissolved, the mixture was diluted with H2O to 1000 L and an additional centrifugation was performed. The supernatant of the iCLIP lysis buffer (1 mL) and the MSB lysis buffer (1 ml) were combined and added to the antibody-coupled beads, which were then rotated at 4C for 2 h (final urea concentration 175 mM). The beads were then washed with high-salt washing buffer (50mM Tris-HCL pH 7.4, 1M NaCl, 1% NP-40, 0.1% SDS, 0.5% sodium deoxycholate). After the first round of washes, the library was split into 10%, which were radioactively labelled (according to the basic iCLIP protocol), and 90%, which proceed through 3 adapter addition, an additional phosphorylation (0.2 l PNK, 0.4 l cold ATP (1mM), 0.4 l 10x PNK buffer, 3 l water) and a 5 marker ligation (6 l water, 5 l 4X ligation buffer, 2 l RNA ligase, 1 l RNasin, 2 l 5 marker (100 M), 4 l PEG400). The sequence of the 5 marker is CAGUCCGACGAUC, which corresponds to the Illumina short RNA 5 Adapter (RA5), part #15013205; this sequence is not complementary to the primers used for amplification of iCLIP cDNA libraries (Huppertz et al., 2014). We then produced sequence reads of 120 nt using the Illumina HiSeq platform for eIF4A3 iCLIP. The rest of the protocol was carried out as previously described (Huppertz et al., 2014). Huppertz, I., Attig, J., D'Ambrogio, A., Easton, L.E., Sibley, C.R., Sugimoto, Y., Tajnik, M., Konig, J., and Ule, J. (2014). iCLIP: protein-RNA interactions at nucleotide resolution. Methods 65, 274-287.

Project description:We sequenced pollen DNA from Col x Ler F1 hybrid plants using Nanopore long-read sequencing and identified crossover sites with COmapper. To purify pollen grains from F1 hybrid plants and extract their genomic DNA, whole inflorescences of hybrid plants were collected and transferred to ice-cold 10% (w/v) sucrose in a 50-ml tubes. Flowers were homogenized with a blender (Tefal, BL3051KR), filtered through 80-μm nylon mesh, and centrifuged at 350g for 10 min at 4 °C. The supernatant was discarded and the pellet was resuspended in 10% (w/v) sucrose, filtered through a 40-μm cell strainer, and centrifuged at 100g for 10 min at 4°C. The purified pellet was frozen in liquid nitrogen and ground with a mortar and pestle. The pellet powder was resuspended in CTAB buffer (1% [w/v] CTAB, 50 mM Tris-HCl pH 8.0, 0.7 M NaCl, 10 mM EDTA). Proteinase K (NEB, P8107S) and RNase A (20 mg/ml, Roche, 10109142001) were added to a final concentration of 80 U/ml and 20 μg/ml, respectively. To check pollen disruption, a 1-μl sample was diluted in a 1:10 ratio with 10% (w/v) sucrose and observed under a stereomicroscope (Leica, M165 FC). Then, 500 μl of the resuspended pellet was transferred to a 1.5-ml tube, to which an equal volume of 25:24:1 (v/v/v) phenol:chloroform:isoamylalcohol (Sigma, 77617) was added, and the mixture was homogenized by gentle manual shaking. The tube was centrifuged at 15,000g for 10 min at 4°C, and 400 μl of supernatant was transferred into a new tube, to which a Nanobind disk (Nanobind® Plant Nuclei Kit RT, PacBio, 102-302-000) was added. The tube containing the Nanobind disk was gently mixed, 400 μl isopropanol was added, and the tube was slowly inverted and incubated for 2 h at room temperature on a rotator set at 7 rpm. A magnetic rack (DynaMagTM-2, Invitrogen, 12321D) was used to separate the disk and the supernatant, and the supernatant was removed. PW1 buffer in the Nanobind Plant Nuclei Kit was used for washing the disk twice and the supernatant was removed completely by centrifugation. Then, 150 μl EB+ buffer was added onto the disc and gently tabbed and incubated overnight at room temperature. The supernatant was transferred to a new 1.5-ml tube and stored at −20°C. The DNA was quantified using a Qubit dsDNA Broad Range assay kit (Thermo Fisher, Q32853) and integrity checked by gel electrophoresis. Nine micrograms of gDNA from pollen was used to construct a nanopore long-read sequencing library using a Ligation Sequencing Kit V14 (Nanopore, SQK-LSK114). The libraries were sequenced using a PromethION platform (BGI, Hong Kong).

Dataset Information

BRD4 regulates the osteogenic differentiation of MSCs

Publications

Super enhancers targeting ZBTB16 in osteogenesis protect against osteoporosis.

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