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BaM-seq and TBaM-seq, highly multiplexed and targeted RNA-seq protocols for rapid, low-cost library generation from bacterial samples [TBaM-seq]

ABSTRACT: Here, we report the development of two RNA-seq library preparation protocols that increase the throughput and decrease the cost of converting RNA to cDNA libraries compatible for sequencing on high-throughput platforms. BaM-seq allows for early barcoding of samples such that many biological samples can be processed simultaneously. TBaM-seq allows for enrichment of target RNAs to decrease the required sequencing depth. Both methods are able to accurately measure gene expression changes with high technical reproducibility and agreement with gold standard, lower throughput approaches.

ORGANISM(S): Bacillus subtilis

PROVIDER: GSE206424 | GEO | 2023/02/01

REPOSITORIES: GEO

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BaM-seq and TBaM-seq, highly multiplexed and targeted RNA-seq protocols for rapid, low-cost library generation from bacterial samples [BaM-seq]

Project description:Here, we report the development of two RNA-seq library preparation protocols that increase the throughput and decrease the cost of converting RNA to cDNA libraries compatible for sequencing on high-throughput platforms. BaM-seq allows for early barcoding of samples such that many biological samples can be processed simultaneously. TBaM-seq allows for enrichment of target RNAs to decrease the required sequencing depth. Both methods are able to accurately measure gene expression changes with high technical reproducibility and agreement with gold standard, lower throughput approaches.

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BaM-seq and TBaM-seq, highly multiplexed and targeted RNA-seq protocols for rapid, low-cost library generation from bacterial samples [BaM-seq]

Project description:BaM-seq and TBaM-seq, highly multiplexed and targeted RNA-seq protocols for rapid, low-cost library generation from bacterial samples [BaM-seq]

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BaM-seq and TBaM-seq, highly multiplexed and targeted RNA-seq protocols for rapid, low-cost library generation from bacterial samples.

Project description:The ability to profile transcriptomes and characterize global gene expression changes has been greatly enabled by the development of RNA sequencing technologies (RNA-seq). However, the process of generating sequencing-compatible cDNA libraries from RNA samples can be time-consuming and expensive, especially for bacterial mRNAs which lack poly(A)-tails that are often used to streamline this process for eukaryotic samples. Compared to the increasing throughput and decreasing cost of sequencing, library preparation has had limited advances. Here, we describe bacterial-multiplexed-seq (BaM-seq), an approach that enables simple barcoding of many bacterial RNA samples that decreases the time and cost of library preparation. We also present targeted-bacterial-multiplexed-seq (TBaM-seq) that allows for differential expression analysis of specific gene panels with over 100-fold enrichment in read coverage. In addition, we introduce the concept of transcriptome redistribution based on TBaM-seq that dramatically reduces the required sequencing depth while still allowing for quantification of both highly and lowly abundant transcripts. These methods accurately measure gene expression changes with high technical reproducibility and agreement with gold standard, lower throughput approaches. Together, use of these library preparation protocols allows for fast, affordable generation of sequencing libraries.

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Transcription profiling of Drosophila ovaries from bam mutant and stwl bam double mutant females

Project description:Abstract: During Drosophila oogenesis, germline stem cell (GSC) identity is maintained largely by preventing the expression of factors that promote differentiation. This is accomplished via the activity of several genes acting either in the GSC or its niche. The translational repressors, Nanos and Pumilio, act in GSCs to prevent differentiation, likely by inhibiting translation of early differentiation factors, while niche signals prevent differentiation by silencing transcription of the differentiation factor Bam. We have found that the DNA-associated protein Stonewall (Stwl) is also required for GSC maintenance. stwl is required cell-autonomously; clones of stwl- germ cells were lost by differentiation, and ectopic Stwl caused an expansion of GSCs. stwl mutants acted as Suppressors of Variegation, indicating stwl normally acts in chromatin-dependent gene repression. In contrast to several previously described GSC maintenance factors, Stwl likely functions epigenetically to prevent GSC differentiation. Stwl-dependent transcriptional repression does not target bam, but rather Stwl represses the expression of many genes, including those that may be targeted by Nanos/Pumilio translational inhibition. Experiment Overall Design: Genetic variation comparison between germ cells of Drosophila ovaries from bam mutant and stwl bam double mutant females. Six total samples were analyzed, with each genotype performed in triplicates.

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