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| Status |
Public on Apr 02, 2021 |
| Title |
Pombe_NonDSN_rep2 |
| Sample type |
SRA |
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| Source name |
Pombe_NonDSN
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| Organism |
Schizosaccharomyces pombe |
| Characteristics |
strain background: h-
parental strain background: Schizosaccharomyces pombe h-
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| Treatment protocol |
For cycloheximide (CHX) treatment, CHX was added to final 0.1 mg/mL to the medium and incubated for 5 min at 30°C for S. cerevisiae and 10 min for S.pombe. For glucose deprivation, cells were shifted to pre-warmed YP (lacking glucose) and then grown at 30°C for 5 minutes and 15 minutes prior to harvesting. For glucose re-addition, cultures with already 15 min glucose deprivation were shifted to YPD for 15 min at 30°C. For early exponential phase, strains were grown at 30°C from an initial OD600~0.05 to a final OD600 of 0.3. To reach stationary stage, S. cerevisiae strains were grown during 60h in YPD.
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| Growth protocol |
S. cerevisiae grown in YPD at 30°C. S.pombe grown in YES at 30°C
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| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was isolated by the standard phenol: chloroform method, and DNA was removed by DNase I treatment. The amount of RNA was quantified in a Nanodrop 2000 Spectrophotometer (Thermo Scientific) and RNA integrity was checked by agarose gel electrophoresis.
For HT5Pseq libraries construction, we used 6 μg of DNA-free total RNA. Samples were directly subjected to RNA ligation. The treated RNA samples were incubated with 100 μM RNA/RNA rP5_RND oligo (final 10 μM) 2h at 25°C with 10 Units of T4 RNA ligase 1 (NEB). Ligated RNA was purified with RNAClean XP (Beckman Coulter), according to the manufacturer’s instructions. RNA was reverse transcribed with Superscript II (Life Technologies) and primed with Illumina PE2 compatible oligos containing random hexamers (20 μM) and oligo-dT (0.05 μM). Reverse transcription reaction was incubated for 10 min at 25°C, 50 min at 42°C and heat inactivation for 15 min at 70°C. To deplete RNA in RNA/cDNA hybrid after reverse transcription, we used sodium hydroxide (40 mM) for incubation 20 min at 65°C and then neutralized with Tris-HCl, pH =7.0 (40 mM). For DSN (Duplex-specific nuclease) based rRNA depletion, we used a mixture of probes targeted the 18S rDNA, 25S rDNA and 5.8S rDNA. The probes were designed to occupy the whole ribosomal RNA regions with consecutive 25-30nt long unmodified DNA oligos. The hybridization of probes (2 μM each) with cDNAs were incubated at 68 °C for 2 minutes before adding pre-warmed DSN buffer mix with 1 Units of DSN enzyme (Evrogen). The reaction then performed at 68 °C for 20 minutes. To inactive DSN enzyme, we added 2X DSN stop solution and incubate 10 min at 68 °C. The final PCR amplification was used 2X Phusion High-Fidelity PCR Master Mix with HF Buffer (NEB) and final 0.1 μM of PE1.0 and corresponding multiplex PE2.0_MTX . The program followed this: 30s 98°C; 15 cycles (20s 98°C; 30s 65°C; 30s 72°C); 7min 72°C. Libraries were size selected using 0.7x-0.9x (v/v) AMpure XP beads (Beckman Coulter) to final 200-500 bp and sequenced by NextSeq 500/550 using 60 sequencing cycles for Read 1 and 15 cycles for Read 2. 5PSeq methods was performed as previously described (PMID: 26820793). 6µg of total RNA was used as input. In brief a RNA oligo (rP5_RND) containing an Illumina adaptor and unique molecular identifiers (UMI) was ligated to the intermediates of mRNA co-translation degradation (5’P). Ribosomal RNA was depleted using Ribo-Zero Magnetic Gold Kit (Illumina).Libraries were PCR amplified (15 cycles). Ampure beads size selected libraries with an average length of 451 nt were sent for sequencing (llumina NextSeq 500 instrument).
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
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| Description |
Pombe-NonDSN_2
HT-5Pseq without DSN treatment in S.pombe
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| Data processing |
library strategy: RNA-Seq (5PSeq)
Base-calling was done using bcl2fastq v2.20.0.422 and for demultiplex, we allowed one mismatch in index1 and one mismatch in index2
For HT-5Pseq data, 3’-sequencing adaptor (-AGATCGGAAGAGCACACGTCTGAACTCCAGTCA) trimming was applied to 5’ends of reads using cutadapt V1.16. The 8-nt random barcodes on the 5’ ends of reads were extracted and added to the header of fastq file as the UMI using UMItools (v0.5.4). Reads were mapped to the reference genome (SGD R64-1-1 for S. cerevisiae genome, ASM294v2.20 for S.pombe genome) separately by star/2.7.0 with the parameter --alignEndsType Extend5pOfRead1 to exclude soft-clipped bases on the 5’ end. To calculate the fraction of rRNA, tRNA, snRNA snoRNA and mRNA in library compositions,the stepwise alignment was performed by corresponding index generated by star/2.7.0. Duplicated 5’ends of read introduced by PCR during library preparation were removed based on random barcodes sequences using UMItools (v0.5.4).
For The distribution of nucleotide position of 5’ends were performed using Fivepseq package , including relative to start, stop codon and codon specific pausing. Specifically, the unique 5’mRNA reads in biological samples were summed up and normalized to reads per million (rpm). Then the relative position of 5’mRNA reads to all codons of all ORF were summed at each position. The metagene plot was showed as the sum value versus the relative distance from respective codon.
Genome_build: Saccharomyces cerevisiae R64-1-1 , Schizosaccharomyces pombe ASM294v2
Supplementary_files_format_and_content: Bedgraph files for UMI collapsed reads for positive and negative strand. Reads were collapsed to the first 5´nucleotide.
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| Submission date |
Jun 12, 2020 |
| Last update date |
Apr 03, 2021 |
| Contact name |
Vicent Pelechano |
| E-mail(s) |
vicente.pelechano.garcia@ki.se
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| Organization name |
ScilifeLab - Karolinska Institutet
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| Department |
MTC
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| Street address |
Nobels väg 16
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| City |
Solna |
| ZIP/Postal code |
SE-17177 |
| Country |
Sweden |
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| Platform ID |
GPL20584 |
| Series (1) |
| GSE152375 |
Development of High-throughput 5Pseq and detection of ribosome stalls at termination level in Saccharomyces cerevisiae |
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| Relations |
| BioSample |
SAMN15223828 |
| SRA |
SRX8537884 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM4613704_YES_NonDSN_Spombe_2_R1_negative.bedGraph.gz |
301.3 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM4613704_YES_NonDSN_Spombe_2_R1_positive.bedGraph.gz |
311.8 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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