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| Status |
Public on Dec 21, 2022 |
| Title |
dTAGSET1AB_RNAseq_Rep2_2hr |
| Sample type |
SRA |
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| Source name |
Mouse embryonic stem cells with Drosophila SG4 cells spike-in, dTAG-SET1A/B cells depleted of dTAG-SET1A and dTAG-SET1B
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| Organisms |
Drosophila melanogaster; Mus musculus |
| Characteristics |
cell line: dTAG-SET1A/B
condition: Mouse embryonic stem cells with Drosophila SG4 cells spike-in, dTAG-SET1A/B cells depleted of dTAG-SET1A and dTAG-SET1B
treatment: 100nM dTAG13
treatment time: 2hr
spike-in reference organism: Drosophila melanogaster
spike-in cell line: SG4
protocol: calibrated RNA-seq
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| Treatment protocol |
Cell lines expressing dTAG fusion proteins were treated with 100 nM dTAG-13 (produced by Behnam Nabet and Nathanael Gray1 or Carole Bataille and Angela Russell) to induce protein depletion.
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| Growth protocol |
Mouse embryonic stem cells were grown in Dulbecco’s Modified Eagle Medium (Thermo Fisher Scientific) supplemented with fetal bovine serum (FBS, 15% Biosera or 10% Sigma), 1x non-essential amino acids (Thermo Fisher Scientific), 2 mM L-glutamine (Thermo Fisher Scientific), 1x penicillin/streptomycin (Thermo Fisher Scientific), 0.5 mM beta-mercaptoethanol (Thermo Fisher Scientific), and 10 ng/ml leukaemia inhibitory factor (produced in-house). ESCs were grown on gelatinised plates at 37⁰C and 5% CO2.
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| Extracted molecule |
total RNA |
| Extraction protocol |
ESCs (~106) were counted and mixed with ¼ of the number of SG4 Drosophila cells in PBS. RNA was extracted from cells using TRIzol reagent, according to the manufacturer’s protocol (Thermo Fisher Scientific). gDNA contamination was depleted using TURBO DNA-free Kit (Thermo Fisher Scientific) and quality of RNA was assessed using 2100 Bioanalyzer RNA 6000 Pico kit (Agilent). 900 ng RNA was depleted of rRNA using the NEBNext rRNA Depletion kit (NEB).
RNA-seq libraries were prepared from an equal amount of ribo-depleted RNA using the NEBNext Ultra II Directional RNA Library Prep kit, including 2.5-4 min fragmentation at 94⁰C (NEB).
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina NextSeq 500 |
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| Data processing |
Reads that aligned to the mm10 and dm6 rDNA genomic sequences (GenBank: BK000964.3 and M21017.1) were first identified using Bowtie2 with ‘-very-fast’, ‘-no-mixed’ and ‘-no-discordant’ options) and discarded. Unmapped reads were then aligned to the concatenated mm10 and dm6 genomes using STAR. To improve mapping of intronic sequences, reads that failed to map using STAR were aligned using Bowtie2 with ‘-sensitive-local’, ‘-no-mixed’ and ‘-no-discordant’ options. Uniquely aligned reads from the last two steps were combined for further analysis and PCR duplicates were removed using Sambamba.
To internally calibrate RNA-seq, we spiked-in a fixed number of Drosophila SG4 to each experimental sample. For data visualisation, mm10 reads were randomly subsampled using factors that reflected the total number of dm6 reads in each sample.
Read counts from biological triplicates or quadruplicates were determined using custom scripts utilising SAMTools for a custom-built, non-redundant mm10 gene set. Briefly, mm10 refGene genes were filtered to remove very short genes with poor sequence mappability and highly similar transcripts which resulted in the final set of 20,633 genes. Raw mm10 reads prior to spike-in normalisation were used for read counts quantitation for differential expression analysis.
The final set of 20,633 genes were used for differential analysis using a custom R script adapting DESeq2 (Love et al., 2014) for spike-in calibrated RNA-seq data. To incorporate spike-in calibration into this analysis, read counts for the spike-in genome at a control set of intervals were supplied to calculate DESeq2 size factors which were then used for DESeq2 normalisation of raw mm10 read counts. A set of unique dm6 refGene genes was used for spike-in normalisation of RNA-seq. For a change to be called significant, we applied a threshold of p-adj < 0.05 and fold change > 1.5.
Assembly: mm10, dm6
Supplementary files format and content: Stranded bigWig files were generated using genomeCoverageBed from BEDTools representing genome coverage of merged spike-in normalised biological replicates.
Supplementary files format and content: DESeq2 results tables for differential gene expression analysis containing normalised read counts, raw and shrunk log2-fold changes (LFC), and statistical significance levels for a non-redundant set of mm10 refGene genes.
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| Submission date |
Mar 30, 2022 |
| Last update date |
Dec 21, 2022 |
| Contact name |
Emilia Dimitrova |
| E-mail(s) |
emilia.dimitrova@bioch.ox.ac.uk
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| Organization name |
University of Oxford
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| Department |
Biochemistry
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| Lab |
Rob Klose
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| Street address |
South Parks Road
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| City |
Oxford |
| ZIP/Postal code |
OX1 3QU |
| Country |
United Kingdom |
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| Platform ID |
GPL25537 |
| Series (2) |
| GSE199803 |
A CpG island-encoded mechanism protects genes from premature transcription termination [SET1_RNAseq] |
| GSE199805 |
A CpG island-encoded mechanism protects genes from premature transcription termination |
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| Relations |
| BioSample |
SAMN27110519 |
| SRA |
SRX14673738 |
| 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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