Project description:Here, we investigate the extent to which individual cells within the total bone marrow (tot-BM) and the lineage negative (Lin-neg) populations exhibit this isoform diversity and whether distinct subpopulations in tot-BM and Lin-neg cells intersect based on transcript isoform usage. We extracted healthy donor human bone marrow tissues from discarded harvesting filters. From this total bone marrow cell preparation (tot-BM) we enriched for lineage-negative progenitor cells (Lin-neg) by magnetic selection as described earlier (Deslattes Mays et al. 2019). We then analyzed total and Lin-neg cell populations by droplet-based single cell RNA sequencing (10xGenomics). In order to increase the number of mRNA molecules detectable per cell we reduced the cell input to approximately 500 cells for each experiment. After single-cell selection, the barcoded cDNA libraries were equally divided and each pool was analyzed in parallel by short-read sequencing (Illumina) and by single-molecule real-time (SMRT) full-length RNA sequencing (Pacific Biosciences).
2023-04-06 | GSE181160 | GEO
Project description:Single Molecule Real-Time DNA sequencing of HLA genes
Project description:In this study, we provided the first genome-wide, base pair-resolution map of 6mA in Tetrahymena by applying single-molecule real-time (SMRT) sequencing.
2017-10-11 | GSE96521 | GEO
Project description:Single Molecule Real-Time (SMRT) of transcriptome in sugarcane treated with low-potassium
Project description:6mA-DNA-IP-Seq and sequencing of Arabidopsis. Our DeepM6A model was trained on the 6mA sites indentified by the single-molecule real-time (SMRT) sequencing. To validate the robustness of the DeepM6A model, we applied an independent DNA-6mA-IP for A. thaliana, and predicted scores of peaks by using the trained DeepM6A model.
Project description:Six bacterial genomes, Geobacter metallireducens GS-15, Chromohalobacter salexigens, Vibrio breoganii 1C-10, Bacillus cereus ATCC 10987, Campylobacter jejuni subsp. jejuni 81-176 and Campylobacter jejuni NCTC 11168, all of which had previously been sequenced using other platforms were re-sequenced using single-molecule, real-time (SMRT) sequencing specifically to analyze their methylomes. In every case a number of new N6-methyladenine (m6A) and N4-methylcytosine (m4C) methylation patterns were discovered and the DNA methyltransferases (MTases) responsible for those methylation patterns were assigned. In 15 cases it was possible to match MTase genes with MTase recognition sequences without further sub-cloning. Two Type I restriction systems required sub-cloning to differentiate their recognition sequences, while four MTases genes that were not expressed in the native organism were sub-cloned to test for viability and recognition sequences. No attempt was made to detect 5-methylcytosine (m5C) recognition motifs from the SMRT sequencing data because this modification produces weaker signals using current methods. However, all predicted m6A and m4C MTases were detected unambiguously. This study shows that the addition of SMRT sequencing to traditional sequencing approaches gives a wealth of useful functional information about a genome showing not only which MTase genes are active, but also revealing their recognition sequences. Examination of the methylomes of six different strains of bacteria using kinetic data from single-molecule, real-time (SMRT) sequencing on the PacBio RS.
Project description:Single Molecule Real Time (SMRT) sequencing was utilized to map the genome-wide m6A methylation pattern in B. mayonii. Consensus modified motifs were identified in wildtype B. mayonii as well as clonal isolates lacking plasmids that encode predicted methyltransferases. Two conserved m6A modification motifs were identified, and were fully attributable to the presence of specific methyltransferases.