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| Status |
Public on Apr 01, 2022 |
| Title |
Mapping Human Transient Transcriptomes Using Single Nucleotide Resolution 4sU Sequencing (SNU-Seq) |
| Organism |
Homo sapiens |
| Experiment type |
Expression profiling by high throughput sequencing
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| Summary |
Genomes are pervasively transcribed leading to stable and unstable transcripts that define functional regions of genomes and contribute to cellular phenotypes. Defining comprehensive nascent transcriptomes is pivotal to understand gene regulation, disease processes, and the impact of extracellular signals on cells. However, currently employed methods are laborious, technically challenging and costly. We developed single-nucleotide resolution 4sU-sequencing (SNU-Seq), involving pulse labelling, biotinylation and direct isolation of nascent transcripts. Artificial poly-(A)-tailing of the 3’ most nucleotide of nascent transcripts ensures oligo-d(T) primer-based library preparation and sequencing using commercial 3’ RNA-Seq kits. We show that SNU-Seq is a cost-effective new method generating even read profiles across transcription units. We used SNU-Seq to identify transcription elongation parameters, to map usage of polyadenylation (PAS) sites and novel enhancers. Remarkably, 4sU labelled nascent RNA accumulates short ~100nt transcripts that map to the 5’ end of genes. We show that isolation of these short nascent RNA and sequencing the 5’ and 3’ ends using size-selected SNU-Seq (ssSNU-Seq) provides highly sensitive annotations of mapped and novel TSSs, promoter-proximal pause/termination sites. Thus, SNU-seq and ssSNU-seq combined yield comprehensive transcriptomics data at low cost with high spatial and temporal resolution.
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| Overall design |
To develop SNU-Seq and size-selected SNU-Seq for measuring nascent transcription at high resolution.
There are 3 different sample types reported here; SNU-seq, size-selected SNU-seq, and TT-seq. There are 11 different samples. All samples are from HEK293 cell and are spike-in controlled with 3 thiolablled 1kb long fragments from yeast with a 42% GC content. TT-seq samples are a time series - 10,15, 20 mins labelling with 4sU with 1 reapeat (samples 4 to 9) or cells untreated or treated with alpha amanitin during a 5 min pulse label with 4sU (samples 10 and 11). For samples 4 to 11 libraries were prepared using the NEBNext II Ultra Directional RNA library and sequenced on the Illumina NextSeq 500 platform using the High-Output Nextseq 500 kit. For each sample 4 to 9 there are 4 processed files (two repeats (A and B) and reads on the forward (F) and reverse (R) strand) and 16 raw samples per time point with R1 and R2 series representing the paired-ends. For samples 10 and 11 there is one repeat, 2 processed files and 8 raw files per sample with R1 and R2 representing the paired ends. SNU-seq samples were thiolabelled for 10 minutes with 4sU and after adding a polyA track to the 3' end, sequenced from the 3' polyA tail using the Quant-Seq Lexogen 3' mRNA kit on the Ion Proton platform. There are two raw data files representing two repeats (17 and 18). There are six processed files representing the merged data on each strand for the full SNU-seq read out primed from the artifical polyA tail, the 3 prime read on each strand (SNU-seq readout) and the 3 prime read after removal of internal polyA tracks >8nts. Size-selected SNU-seq samples (ssSNU-seq) were labelled as for the SNU-seq but the polyadenylation step was omitted. Instead 50-100bp thiolabelled RNA fragments were selected from the nascent thiolabelled RNA using a gel, adaptors ligated to the 5' or 3' ends and sequenced using an NEBNext Small RNA library kit on the Illumina platform.There are 4 raw data files and four processed files, two for the 5' ends and two for the 3' ends on each strand. In summary there are 24 processed files and 70 raw data files.
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| Contributor(s) |
Lorenz P, Lamstaes A, Xi S, Murray S, Mellor J |
| Citation missing |
Has this study been published? Please login to update or notify GEO. |
| Submission date |
Jul 01, 2021 |
| Last update date |
Apr 02, 2022 |
| Contact name |
Jane Mellor |
| E-mail(s) |
Jane.mellor@bioch.ox.ac.uk
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| Phone |
07810544459
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| Organization name |
University of Oxford
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| Department |
Biochemistry
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| Lab |
Mellor
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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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| Platforms (2) |
| GPL17303 |
Ion Torrent Proton (Homo sapiens) |
| GPL18573 |
Illumina NextSeq 500 (Homo sapiens) |
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| Samples (10)
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| Relations |
| BioProject |
PRJNA746479 |
| SRA |
SRP328288 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSE179306_RAW.tar |
3.1 Gb |
(http)(custom) |
TAR (of BEDGRAPH, BW) |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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