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Bulk RNA‑seq profiling of CD69‑overexpressing and control NB4 cells

ABSTRACT: Total RNA was extracted from CD69-overexpressing NB4 cells and matched control NB4 cells using TRIzol (Thermo Fisher, Cat#15596018). RNA quantity and purity were evaluated using NanoDrop ND-1000, and RNA integrity was confirmed on an Agilent Bioanalyzer 2100, with samples meeting the criteria of concentration >50 ng/µL, total RNA >1 µg, and RIN >7.0. Polyadenylated mRNA was enriched using Dynabeads Oligo(dT) (Thermo Fisher, Cat#25-61005) with two rounds of purification. Fragmentation was performed using the NEBNext Magnesium RNA Fragmentation Module (NEB, Cat#E6150S) at 94°C for 5–7 minutes. First-strand cDNA was synthesized with SuperScript II (Invitrogen, Cat#1896649), and second-strand synthesis used DNA Polymerase I (NEB, Cat#M0209) and RNase H (NEB, Cat#M0297) incorporating dUTP (Thermo Fisher, Cat#R0133) to generate strand-specific libraries. Libraries were end-repaired, A-tailed, ligated to adapters, and size-selected (~300 bp ± 50 bp), followed by UDG treatment and PCR amplification. Sequencing was performed on an Illumina NovaSeq 6000 (PE150). Resulting FASTQ files and processed gene expression matrices are included in this submission.

ORGANISM(S): Homo sapiens

PROVIDER: GSE313879 | GEO | 2026/03/31

REPOSITORIES: GEO

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	GSE313879_gene_count_matrix.txt.gz	Txt

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Project description:The Recombinant human bone morphogenetic protein 2 (rhBMP-2) and the human bone marrow mesenchymal stromal cells (hBM-MSCs) have been thoroughly studied with research and translational bone regenerative purposes. rhBMP-2 induces in vivo the endochondral ossification and bone formation processes, while hBM-MSCs are considered its target, bone forming cells. The in vitro ability of rhBMP-2 to induce hBM-MSC osteogenic differentiation has been previously tested. However, bone formation implies mesenchymal cell differentiation to multiple phenotypes, including chondrogenesis, adipogenesis and also osteogenesis. In this article, we wondered whether rhBMP-2 may drive not only osteogenic, but also multilineage differentiation of hBM-MSCs, both in vivo and in vitro. aiming to model all the differentiation processes observed in the rhBMP-2 induced in vivo bone formation. First, the rhBMP-2 and hBM-MSCs were tested in an in vivo implantation model to assess their ability to induce mature bone formation and mutilineage differentiation of implanted cells. Then, hBM-MSCs were in vitro treated with rhBMP-2 at short- and long-term cell culture periods, alone or in combination with osteogenic, adipogenic and chondrogenic media, aiming to define the role of rhBMP-2 in these differentiation processes. Data indicate that hBM-MSCs respond to rhBMP-2 at short term, but fail to differentiate in long term cultures, which was linked to the induction of rhBMP-2 target genes DKK1, HEY-1 and SOST osteogenesis inhibitors. However, when rhBMP-2 is used in combination with other differentiation signals in in vitro long term cell cultures, the mentioned negative feedback loop mechanism disappears and rhBMP-2 acts as potentiator of multilineage differentiation, not only osteogenesis, but also adipogenesis and chondrogenesis. Altogether, data indicate rhBMP-2 alone is unable to induce hBM-MSC terminal differentiation in in vitro settings, but synergizes with other signals to potentiate multiple differentiation phenotypes. When using rhBMP-2 and hBM-MSCs for translational bone formation applications, they should be considered that these regenerative agents are acting in a system together with other signals, triggering multiple phenotypes depending on the signaling environment, to yield the formation of mature bone and bone marrow tissues. In vivo implantation assay and sample testing: All animals were maintained and handled in accordance with the Guidelines for Accommodation and Care of Animals (European Convention for Protection of Vertebrate Animals Used for Experimental and Other Scientific Purposes) and internal guidelines. Experimental procedures were approved by the ethical committee and local competent authorities (Project number PRO-AE-SS-171). All animals were housed in ventilated cages and fed with standard diet ad libitum. E. coli produced recombinant BMP-2 (rhBMP-2) was obtained from commercial providers (Noricum SL, Cat.# BMP2). RNA Sequencing and data processing : The quantity and quality of the RNAs were evaluated using Qubit RNA HS Assay Kit (Thermo Fisher Scientific, Cat.# Q32855) and Agilent RNA 6000 Nano Chips (Agilent Technologies, Cat.# 5067-1511), respectively. Sequencing libraries were prepared following “TruSeq Stranded mRNA Sample Preparation Guide (Part # 15031058 Rev. E)” using the “TruSeq Stranded mRNA Library Prep” kit (Illumina Inc. Cat. # 20020594) and TruSeq RNA CD Index Plate (96 Indexes, 96 Samples) (Illumina Inc. Cat. # 20019792). Starting from 700 ng of total RNA, mRNA was purified, fragmented and primed for cDNA synthesis. cDNA first strand was synthesized with SuperScript-II Reverse Transcriptase (Thermo Fisher Scientific, Cat. # 18064-014) for 10 min at 25°C, 15 min at 42°C, 15 min at 70°C and pause at 4°C. cDNA second strand was synthesized with Illumina reagents at 16°C for 1 hour. Then, A-tailing and adaptor ligation were performed. Finally, enrichment of libraries was achieved by PCR (30 sec at 98°C, 15 cycles of 10 sec at 98°C, 30 sec at 60°C, 30 sec at 72°C, 5 min at 72°C and pause at 4°C). Afterwards, libraries were visualized on an Agilent 2100 Bioanalyzer using Agilent High Sensitivity DNA kit (Agilent Technologies, Cat. # 5067-4626 ) and quantified using Qubit dsDNA HS DNA Kit (Thermo Fisher Scientific, Cat. # Q32854).

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Bulk RNA‑seq profiling of CD69‑overexpressing and control NB4 cells

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