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| Status |
Public on Nov 26, 2024 |
| Title |
PNUTSdTAG_TTseq_dTAG_rep3 |
| Sample type |
SRA |
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| Source name |
E14 PNUTS-dTAG
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| Organisms |
Drosophila melanogaster; Mus musculus |
| Characteristics |
cell line: E14 PNUTS-dTAG
cell type: embryonic stem cell line
tag: 3xT7-2xStrepII-dTAG
rescue: none
spike-in cell_line: SG4
treatment: dTAG13
treatment time: 2h
additional treatment: none
additional treatment_time: 0h
labelling: 500 µM 4-thiouridine
labelling time: 15 min
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| Treatment protocol |
Cell lines expressing dTAG fusion proteins were treated with 100 nM dTAG13 to induce depletion of PNUTS. For rescue experiments, cells were treated with 1ug/ml DOX to induce the expression of PNUTS WT, PNUTS W401A or PNUTS TND-M and with 100 nM dTAG13 to deplete endogenous PNUTS protein.
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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 |
other |
| Extraction protocol |
cTT-seq was performed largely as described previously (Gregersen et al., 2020). In brief, 9 million ESCs and 3 million Drosophila SG4 cells were labelled with 500 µM 4-thiouridine (4sU, Glentham Life Sciences) for 15 min and harvested into TRIzol reagent. 4sU-labelled mouse and Drosophila cells were mixed and RNA was extracted and depleted from any gDNA carry-over using the Direct-zol RNA Miniprep Plus kit (R2072 Zymo Research) as per the manufacturer’s protocol. An equal quantity of RNA (80-100 µg) was taken into 100 µl nuclease free water and fragmented on ice with 20 µl 1 M NaOH for 20 min. Fragmentation was stopped with 80 μl 1 M Tris, pH 6.8 and the RNA was cleaned up with Micro Bio-Spin P-30 gel columns (Biorad). RNA was biotin-labelled with 50 μl 0.1 mg/ml MTSEA biotin-XX linker (Biotium) with 3 μl biotin buffer (833 mM Tris HCl, pH 7.4, 83.3 mM EDTA) for 30 min at RT. Biotin-labelled RNA was purified with a 1:1 ratio of phenol/chloroform/isoamyl alcohol (Thermo Fisher Scientific). Streptavidin pull-down was performed with the μMACS Streptavidin Kit (Miltenyi Biotec), washing the columns three times with 55 °C pull-down wash buffer (100 mM Tris HCl, pH 7.4, 10 mM EDTA, 1 M NaCl and 0.1% Tween 20) and 3x RT pull down wash buffer. Biotin-labelled RNA was eluted with 100 μl elution buffer (100 mM DTT in nuclease-free water) and cleaned up with the RNeasy MinElute Cleanup kit (QIAGEN), adjusting the amount of ethanol to capture RNA < 200 nucleotides in length.
RNA was quantified using the Qubit RNA HS assay kit and RNA libraries were prepared from 20 ng RNA with the Ultra II Directional RNA library prep kit, as per the manufacturer’s guidelines for rRNA depleted and FFPE RNA (NEB).
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| Library strategy |
OTHER |
| Library source |
other |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
calibrated TT-seq
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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 TT-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 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 TT-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 TT-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.
Library strategy: cTT-seq
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| Submission date |
Jul 04, 2024 |
| Last update date |
Nov 26, 2024 |
| Contact name |
Emilia Dimitrova |
| E-mail(s) |
[email protected]
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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) |
| GSE271484 |
The PNUTS phosphatase complex controls transcription pause release [cTT-seq] |
| GSE271892 |
The PNUTS phosphatase complex controls transcription pause release |
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| Relations |
| BioSample |
SAMN42323092 |
| SRA |
SRX25209242 |
| Supplementary data files not provided |
SRA Run Selector |
| Raw data are available in SRA |
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