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| Status |
Public on May 06, 2020 |
| Title |
5BrU, 4SU, 6SG |
| Sample type |
SRA |
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| Source name |
ERCC Spike-in Mix
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| Organism |
synthetic construct |
| Characteristics |
method protocol: Direct RNA nanopore sequencing (synthetic RNAs)
cell line: -
treatment condition: 5BrU, 4SU, 6SG, IVT
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| Treatment protocol |
Nanopore: K562 cells were kept at low passage numbers (<6) and at optimal densities (3x10^5 - 8x10^5) during all experimental setups. Per biological replicate, K562 cells were diluted 24 h before the experiment was performed (Supplementary Table 1). Per 5EU 60 min sample (6 replicates), cells were incubated at 37 °C, 5 % CO2 for 1 h after a final concentration of 500 µM 5-Ethynyluridine (5EU, Jena Bioscience) was added. Per 5EU 24 h sample (3 replicates), cells were incubated at 37 °C, 5% CO2 for 24 h. 5EU was added 3 times during the 24h incubation, i.e. every 8 hours (0h, 8h, 16h) at a final concentration of 500 µM. Control samples were not labeled (3 replicates). Per 5EU 60 min HS (heat shock) sample (5 replicates), cells were incubated at 42 °C for 5 min (until cell suspension reached 42 °C), and then 5EU was added at a final concentration of 500 µM. Further, heat shock treatments were performed in a water bath (LAUDA, Aqualine AL12) at 42 °C. for 1 h. Temperature was monitored by thermometer. To avoid transcriptional changes by freshly added growth medium, fresh growth medium was added 24 h prior to heat shock treatments 45. Exactly after the labeling duration, cells were centrifuged at 37 °C and 1,500 x g for 2 min.
RNA-seq: Two biological replicates of K562 cells were diluted 24 h before the experiment was performed. Per replicate, 3.6 x 107 cells in growth medium were labeled at a final concentration of 500 µM 4-thio-uracil (4sU, Sigma-Aldrich), and incubated at 37 °C, 5 % CO2 for 5 min. Exactly after 5 min of labeling, cells were harvested at 37 °C and 1,500 x g for 2 min.
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| Growth protocol |
Human K562 erythroleukemia cells were obtained from DSMZ (Cat. # ACC-10). K562 cells were cultured antibiotic-free in accordance with the DSMZ Cell Culture standards in RPMI 1640 medium (Thermo Fisher Scientific) containing 10 % heat inactivated fetal bovine serum (FBS) (Thermo Fisher Scientific), and 1x GlutaMAX supplement (Thermo Fisher Scientific) at 37 °C in a humidified 5 % CO2 incubator. Cells used in this study display the phenotypic properties, including morphology and proliferation rate, that have been described in literature. Cells were verified to be free of mycoplasma contamination using Plasmo Test Mycoplasma Detection Kit (InvivoGen). Biological replicates were cultured independently.
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| Extracted molecule |
total RNA |
| Extraction protocol |
Direct RNA nanopore sequencing library kit (SQK-RNA001, Oxford Nanopore Technologies)
Nanopore: Total RNA was extracted from K562 cells using QIAzol (Quiagen) according to manufacturer’s instructions. Poly(A) RNA was purified from 1 mg of total RNA using the µMACS mRNA Isolation Kit (Milteny Biotec) following the manufacturer’s protocol. The quality of poly(A) RNA selection was assessed using the TapeStation System (Agilent). Poly(A) selected RNAs were subsequently subjected to direct RNA nanopore sequencing library preparation (SQK-RNA001, SQK-RNA002, Oxford Nanopore Technologies) following manufacturer’s protocol with 1000 ng input. All libraries were sequenced on a MinION Mk1B (MIN-101B) for 48 h, unless reads sequenced per second stagnated dramatically.
RNA-seq: Total RNA was extracted from K562 cells using QIAzol according to manufacturer’s instructions except for the addition of 150 ng RNA spike-in mix 25 together with QIAzol. To isolate polyA RNA from 75 µg of total RNA, two subsequent rounds of purification by Dynabeads Oligo (dT)25 (invitrogen) were performed. Purification based on manufacturer’s instructions was performed twice, using 1 mg of Dynabeads Oligo (dT)25 beads for the first round and 0.5 mg for the second round of purification. The quality of polyadenylated RNA selection was assessed using RNA ScreenTape on a TapeStation (Agilent). Sequencing libraries were prepared using the NuGEN Ovation Universal RNA-seq kit according to manufacturer’s instructions. Fragments were amplified by 10 cycles of PCR, and sequenced on an Illumina NextSeq 550 in paired-end mode with 75 bp read length.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
MinION |
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| Description |
Labeled RNA
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| Data processing |
Direct RNA nanopore sequencing data preprocessing of synthetic RNAs. Direct RNA nanopore sequencing reads were obtained for each of the samples (Supplementary Table 1). FAST5 files were base-called using Guppy 3.1.5 (Oxford Nanopore Technologies) with the following parameters: guppy_basecaller -c rna_r9.4.1_70bps_hac.cfg. Direct RNA nanopore sequencing reads were mapped with GraphMap 0.5.2 46 to the synthetic RNA reference sequence with the following parameters: graphmap align --evalue 1e-10. Further data processing was carried out using the R/Bioconductor environment.
Direct RNA nanopore sequencing data preprocessing of human K562 cells. Direct RNA nanopore sequencing reads were obtained for each of the samples (Supplementary Table 1). FAST5 files were base-called using Guppy 3.1.5 (Oxford Nanopore Technologies) with the following parameters: guppy_basecaller -c rna_r9.4.1_70bps_hac.cfg. Direct RNA nanopore sequencing reads were mapped with Minimap2 2.10 47 to the GRCh38/hg38 genome assembly (Human Genome Reference Consortium) with the following parameters: minimap2 -ax splice -k14 --secondary=no. Samtools 1.3.1 48 was used to quality filter SAM files, whereby alignments with MAPQ smaller than 20 (-q 20) were skipped. Further data processing was carried out using the R/Bioconductor environment and custom python scripts.
RNA-seq data preprocessing and antisense bias correction. Paired-end 75 base reads with additional 6 base reads of barcodes were obtained for each of the samples (Supplementary Table 1). Reads were demultiplexed and mapped with STAR 2.3.0 (Dobin and Gingeras 2015) to the hg20/hg38 (GRCh38) genome assembly (Human Genome Reference Consortium). Samtools (Li, Handsaker et al. 2009) was used to quality filter SAM files, whereby alignments with MAPQ smaller than 7 (-q 7) were skipped and only proper pairs (-f2) were selected. Further data processing was carried out using the R/Bioconductor environment. We used a spike-in (RNAs) normalization strategy essentially as described (Schwalb, Michel et al. 2016) to allow observation of antisense bias ratio (ratio of spurious reads originating from the opposite strand introduced by the reverse transcription reaction). Read counts (kij) for spike-ins were calculated using HTSeq (Anders, Pyl et al. 2015). The number of transcribed bases (tbj) for all samples was calculated as the sum of the coverage of evident (sequenced) fragment parts (read pairs only) for all fragments with an inner mate interval not entirely overlapping a Refseq annotated intron (UCSC RefSeq GRCh38) in addition to the sum of the coverage of non-evident fragment parts (entire fragment). The number of transcribed bases (tbj) or read counts (kj) for all features (RefSeq-TUs) were corrected for antisense bias cj as follows using the parameter calculated as described above. RPKs were calculated upon antisense bias corrected read counts (kj) falling into the region of a RefSeq-TU divided by its length in kilobases. Coverages were calculated upon antisense bias corrected number of transcribed bases (tbj) falling into the region of a RefSeq-TU divided by its length in bases.
Genome_build: hg20/hg38 (GRCh38) genome assembly (Human Genome Reference Consortium http://hgdownload.soe.ucsc.edu/downloads.html#human)
Supplementary_files_format_and_content: bigwig files containing the coverage
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| Submission date |
Jan 22, 2020 |
| Last update date |
May 07, 2020 |
| Contact name |
Björn Schwalb |
| E-mail(s) |
bschwal@gwdg.de
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| Organization name |
MPI
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| Street address |
Fassberg 11
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| City |
Göttingen |
| ZIP/Postal code |
37077 |
| Country |
Germany |
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| Platform ID |
GPL25738 |
| Series (1) |
| GSE127890 |
Native molecule sequencing by nano-ID reveals synthesis and stability of RNA isoforms |
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| Relations |
| BioSample |
SAMN13902136 |
| SRA |
SRX7619606 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4279367_maier.cramer.2017.MinION.20170609_1149_labeled_run.coverage.track.minus.bw |
75.8 Kb |
(ftp)(http) |
BW |
| GSM4279367_maier.cramer.2017.MinION.20170609_1149_labeled_run.coverage.track.plus.bw |
89.6 Kb |
(ftp)(http) |
BW |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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