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Depletion of 16S ribosomal RNA improves single-cell RNA-seq of planarians by DASH

ABSTRACT: Single-cell transcriptomics (scRNA-seq) has revolutionized our understanding of cell types and states in various contexts, such as development and disease. Most methodology relies on poly(A) enrichment to selectively capture protein-coding polyadenylated transcripts, intending to exclude ribosomal transcripts that constitute >80% of the transcriptome. However, it is common for ribosomal transcripts to sneak into the library, which can add significant background by flooding libraries with irrelevant sequences. The challenge of amplifying all RNA transcripts from a single cell has motivated the development of new technologies to optimize retrieval of transcripts of interest. This problem is notable in planarians, where we find that 16S ribosomal transcripts were widely enriched (20-80%) across single-cell methods. Therefore, we adapted the Depletion of Abundant Sequences by Hybridization (DASH) to the standard 10X scRNA-seq protocol. We designed single-guide RNAs tiling the 16S sequence for CRISPR-mediated degradation, and subsequently generated untreated and DASH-treated datasets from the same libraries to enable a side-by-side comparison of the effects of DASH. DASH specifically removes 16S sequences without off-target removal of other genes. By assessing the cell barcodes shared by both libraries, DASH-treated cells have consistently higher complexity given the same amount of reads, which allows the detection of a rare cell cluster and more differentially expressed genes. In conclusion, DASH can be easily integrated into existing sequencing protocols and customized to deplete any unwanted transcripts.

ORGANISM(S): Schmidtea mediterranea

PROVIDER: GSE231548 | GEO | 2023/08/07

REPOSITORIES: GEO

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Project description:Comparison between transcriptomes of dash mutants vs WT plants and ectopic expression of 35S::DASH in hairy roots versus empty vector Loss of function: 3 WT plants A17 at 8 days after pollination and 3 dash mutant plants at 8 days afer pollination. Gain of function transformants: 3 transformed plants containing empty vector and 4 transformed plants containing 35S::DASH Total RNA was extracted using a modified cetyltrimethylammonium bromide method (Verdier et al., 2008). 5ug of total RNA from each sample was purified (RNeasy MinElute Cleanup kit; Qiagen) according to the manufacturer?s instructions. RNA was quantified and evaluated for purity using an ND-1000 Nanodrop Spectrophotometer (NanoDrop Technologies) and a Bioanalyzer 2100 (Agilent). The Affymetrix M. truncatula GeneChip Array (Affymetrix) was used for expression analysis during seed development. RNA from three (for dash mutant analysis) and four (for DASH ectopic expression analysis) independent biological replicates were analysed for each time point. Probe synthesis/labelling was carried out from 500 ng of RNA using the GeneChip 3?IVT express kit, according to the manufacturer?s instructions (Affymetrix). Array hybridization, scanning, and data normalization were performed as described by Benedito et al., (2008). Each file from the hybridized Affymetrix array was exported from GeneChip Operating Software version 1.4 (Affymetrix) and imported into Robust Multiarray Average Express (Irizarry et al., 2003) for global normalization. Presence/absence call for each probe set to remove background noise was obtained using dCHIP (Li and Wong, 2001). To identify probe sets differentially expressed in dash mutant vs WT control, the R package Anapuce (J. Aubert, UMR 518 AgroParisTech/INRA) was used. For each probe set, a paired t-test was performed on the log2 expression data from three arrays (3 independent biological repeats for 8 dap data), assuming that the variance of the log expression was the same for all transcripts per genotype. Spots with extreme specific variance, too low or too high, were excluded from the statistical analysis (details on the procedure given in Gagnot et al., 2008). P-values were adjusted by the Benjamini-Hochberg method (Benjamini and Hochberg, 1995), which controls the family-wise error rate.

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Depletion of 16S ribosomal RNA improves single-cell RNA-seq of planarians by DASH

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