Amniota
Ontology highlight
ABSTRACT:
PROVIDER: PRJDB35418 | ENA |
REPOSITORIES: ENA
Similar Datasets
Project description:For analysis of mRNA expression levels, total RNA was harvested from each cell-line in replicate with Trizol™ (Thermo scientific). Total RNA was purified using Direct-zol™ columns according to the manufacturers specifications (Zymo Research). For cDNA synthesis 1 μg of total RNA was process as the T12VN-PAT assay (Jänicke et al., RNA 2012), except that this was adapted for multiplexing on the Illumina MiSeq instrument. We refer to this assay as mPAT for multiplexed PAT. The approach is based on a nested-PCR that sequentially incorporates the Illumina platform’s flow-cell specific terminal extensions onto 3’ RACE PCR amplicons. First, cDNA was generated using the anchor primer mPAT Reverse, next this primer and a pool of 50 gene-specific primers were used in 5 cycles of amplification. Each gene-specific primer had a universal 5’ extension (see supplementary file primers) for sequential addition of the 5’ (P5) Illumina elements. These amplicons were then purified using NucleoSpin columns (Macherey-Nagel), and entered into second round amplification using the universal Illumina Rd1 sequencing Primer and TruSeq indexed reverse primers from Illumina. Second round amplification was for 14 cycles. Note, that each experimental condition was amplified separately in the first round with identical primers. In the second round, a different indexing primer was used for each experimental condition. All PCR reactions were pooled and run using the MiSeq Reagent Kit v2 with 300 cycles (i.e. 300 bases of sequencing) according to the manufacturers specifications. Data were analysed using established bioinformatics pipelines (Harrison et al., RNA 2015)
2016-09-08 | GSE84913 | GEO
Project description:We describe a novel workflow named Barcode Assembly foR Targeted Sequencing, which is a highly sensitive, quantitative, and inexpensive technique for targeted sequencing of transcript cohorts (rBART-Seq) or genomic regions (gBART-Seq) from thousands of bulk samples or single cells in parallel. Multiplexing is based on a simple method that produces extensive matrices of diverse DNA barcodes attached to invariant primer sets, for generating amplicons with dual indices. Here, we used the gBART-Seq for genetic screening of breast cancer patients and identified BRCA mutations with very high precision.
2019-07-07 | GSE130421 | GEO
Project description:We describe a novel workflow named Barcode Assembly foR Targeted Sequencing, which is a highly sensitive, quantitative, and inexpensive technique for targeted sequencing of transcript cohorts (rBART-Seq) or genomic regions (gBART-Seq) from thousands of bulk samples or single cells in parallel. Multiplexing is based on a simple method that produces extensive matrices of diverse DNA barcodes attached to invariant primer sets, for generating amplicons with dual indices. Here, we used the rBART-Seq for RNA quantification, and for the analysis of developmental states of thousands of single human pluripotent stem cells maintained in different media (mTeSR™1, KSR-bFGF, and E8).
2019-07-07 | GSE130422 | GEO
Project description:We describe a novel workflow named Barcode Assembly foR Targeted Sequencing, which is a highly sensitive, quantitative, and inexpensive technique for targeted sequencing of transcript cohorts (rBART-Seq) or genomic regions (gBART-Seq) from thousands of bulk samples or single cells in parallel. Multiplexing is based on a simple method that produces extensive matrices of diverse DNA barcodes attached to invariant primer sets, for generating amplicons with dual indices. Here, we used the rBART-Seq to analyze cell subpopulations that emerge during 72 hours of Wnt/β-catenin pathway activation of H9 hESCs using recombinant Wnt protein (rWnt3a), a small molecule inhibitor of GSK-3 (CHIR99021), or Dox-inducible constitutively active β-catenin (ΔN90).
2019-07-07 | GSE130420 | GEO
Project description:Mitochondrial fatty acid β-oxidation (FAO) is essential for energy production and cellular homeostasis, yet its specific role in sperm function has remained unclear. Through whole-exome sequencing of 673 patients with asthenozoospermia, we identified biallelic TEX44 variants in six individuals, all of whom exhibited defective mitochondrial sheath assembly and impaired sperm motility. Using Tex44 knockout mice, we demonstrated that TEX44 interacts with carnitine palmitoyltransferase 1B (CPT1B) to form a mitochondrial glue, anchoring adjacent mitochondria and facilitating the assembly of the sperm-specific mitochondrial sheath. In vitro, we show that purified TEX44 protein can modulates CPT1B enzymatic activity, limiting the conversion of long-chain fatty acids such as palmitic acid and myristic acid into acyl-carnitines, thereby reducing reactive oxygen species (ROS) production. Loss of TEX44 disrupts this regulatory mechanism, leading to unregulated FAO, excessive ROS generation, and severe oxidative damage to sperm DNA and flagellar structure. Additionally, germ cell-specific Cpt1b knockout mice exhibited phenotypes similar to TEX44 deficiency, including mitochondrial sheath defects and reduced sperm motility. These findings reveal a sperm-specific mechanism by which TEX44 regulates CPT1B activity to balance FAO and ROS generation, providing critical insights into energy metabolism, mitochondrial integrity, and male infertility.
2025-07-14 | MTBLS12397 | MetaboLights
Project description:<p>Mitochondrial fatty acid β-oxidation (FAO) is essential for energy production and cellular homeostasis, yet its specific role in sperm function has remained unclear. Through whole-exome sequencing of 673800 patients with asthenozoospermia, we identified biallelic TEX44 variants in six individuals, all of whom exhibited defective mitochondrial sheath assembly and impaired sperm motility. Using Tex44 knockout mice, we demonstrated that TEX44 interacts with carnitine palmitoyltransferase 1B (CPT1B) to form a mitochondrial glue, anchoring adjacent mitochondria and facilitating the assembly of the sperm-specific mitochondrial sheath. In vitro, we show that purified TEX44 protein can TEX44 modulates CPT1B enzymatic activity, limiting the conversion of long-chain fatty acids such as palmitic acid and myristic acid into acyl-carnitines, thereby reducing reactive oxygen species (ROS) production. Loss of TEX44 disrupts this regulatory mechanism, leading to unregulated FAO, excessive ROS generation, and severe oxidative damage to sperm DNA and flagellar structure. Additionally, germ cell-specific Cpt1b knockout mice exhibited phenotypes similar to TEX44 deficiency, including mitochondrial sheath defects and reduced sperm motility. These findings reveal a sperm-specific mechanism by which TEX44 regulates CPT1B activity to balance FAO and ROS generation, providing critical insights into energy metabolism, mitochondrial integrity, and male infertility.</p>
2025-07-14 | MTBLS12402 | MetaboLights
Project description:The aim of the study was to use microarray for profiling the microbiota in anaerobic digestion process. The probes are ssDNA molecules that are ligated into circular molecules if a complementary target sequence is present in the sample DNA. Ligated probes are PCR amplified with a labeled primer, and the amplicons are hybridized on DNA microarray by tag sequences.
2012-02-06 | E-MEXP-3541 | biostudies-arrayexpress