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| Status |
Public on Feb 09, 2023 |
| Title |
III_C5_S29 |
| Sample type |
SRA |
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| Source name |
bacterial cell
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| Organism |
Salmonella enterica subsp. enterica serovar Typhimurium |
| Characteristics |
cell type: bacterial cell
media: Lennox broth media
od: OD 0.3
condition: MEP
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| Extracted molecule |
total RNA |
| Extraction protocol |
Bacterial RNA was isolated from Salmonella strain SL1344. RNA extraction was performed with 1.8 ml of each in vitro culture using the TRIzol reagent (Invitrogen) according to the manufacturer’s recommendation. RNA quality was checked using Qubit RNA High Sensitivity Assay Kit (Invitrogen) and 2100 Bioanalyzer RNA 6000 Pico/ Nano kit (Agilent Technologies). Prior library preparation, DNAse treatment was performed using DNAse I kit (Thermo Fisher) followed by ribosomal RNA depletion. Ribosomal RNA (rRNA) was depleted using Lexogen's RiboCop META rRNA Depletion Kit protocol according to manufacturer’s recommendation using 100ng total RNA as input per sample.
cDNA obtained from single-cells after MATQseq was further processed for library preparation including ribosomal depletion protocol. Library preparation was done using the Nextera XT DNA Library Preparation Kit (Illumina) including DASH protocol (according to (Prezza et al. 2020) with several modifications). Tagmentation was performed with only ¼ of reaction and 0.5 ng cDNA input according to manufacturer’s recommendations. Index PCR was performed with 13 cycles using IDT for Illumina Nextera DNA Unique Dual Indexes (Illumina). Obtained libraries were purified with AMPure XP beads (Beckman Coulter) at a 1:1 v/v ratio to ensure capturing sRNA-derived cDNA. After QC, up to 12 samples were pooled equimolar for ribosomal depletion. sgRNA/Cas9 complex formation is followed by DASH using the appropriate ratio of cDNA : Cas9 : sgRNA. Cas9 enzyme was inactivated by Proteinase K (15 min at 37 °C). Afterwards, Proteinase K was inactivated by adding PMSF (1 mM final concentration). Depleted cDNA was purified by another round of AMPure XP bead clean-up and used as input for second PCR amplification. 0.5 ng depleted cDNA was used as input for Nextera XT reactions omitting the tagmentation steps. As primers i5 and i7 index-independent primers were used to amplify non-cleaved cDNA products. PCR was done with 13 cycles and clean-up was performed with AMPure XP beads at a 1:1 v/v ratio
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic single cell |
| Library selection |
cDNA |
| Instrument model |
NextSeq 2000 |
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| Description |
III_C5_S29_R1_001
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| Data processing |
Read trimming and quality control of MATQ-seq reads was executed using BBDuk. To efficiently remove primer and adapter sequences located at both ends of a read, we ran BBDuk in a two-pass procedure using the default adapter sequence database augmented with MATQ-seq specific sequences. The first pass focused on the 5’ end, with parameters: minlen=18 qtrim=rl trimq=20 ktrim=l k=17 mink=11 hdist=1 trimpolya=30; while the second pass focused on the 3’ end with parameters: minlen=18 qtrim=rl trimq=20 ktrim=r k=17 mink=11 hdist=1.
Read alignment and counting was performed with Bowtie2 (Langmead & Salzberg 2012) and featureCounts (Liao et al. 2014), allowing a single mismatch and run-in --local mode. BigWig files were generated using deepTools (Ramírez et al. 2014), passing additional parameters: --binSize 5 --normalizeUsing BPM. We have employed the same gene detection method from the original MATQ-seq analyses (Imdahl et al. 2020), requiring a detected gene to have > 5 reads.
DESeq2 (Love et al. 2014) was used for normalization and differential expression analysis, using size factors calculated by the computeSumFactors function in Scran (Lun et al. 2016) and other recommended parameters for DESeq2 single cell analysis, which included using the likelihood ratio test (LRT), useT=TRUE, minmu=1e-6, and minReplicatesForReplace=Inf.
Assembly: NC_016810.1
Supplementary files format and content: Unprocessed count matrix from all samples
Supplementary files format and content: sc_counts.tsv
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| Submission date |
Nov 23, 2022 |
| Last update date |
Feb 09, 2023 |
| Contact name |
Regan Hayward |
| Organization name |
Helmholtz Institute for RNA-based Infection Research
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| Street address |
Josef-Schneider-Straße 2 / D15
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| City |
Wurzburg |
| ZIP/Postal code |
97080 |
| Country |
Germany |
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| Platform ID |
GPL30637 |
| Series (2) |
| GSE218632 |
Improved bacterial single-cell RNA-seq through automated MATQ-seq [scRNA-seq] |
| GSE218633 |
Improved bacterial single-cell RNA-seq through automated MATQ-seq |
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| Relations |
| BioSample |
SAMN31851846 |
| SRA |
SRX18362951 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data are available on Series record |
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