 |
 |
GEO help: Mouse over screen elements for information. |
|
| Status |
Public on Aug 18, 2021 |
| Title |
not4∆_tot02_SC_SP_ACTGGT |
| Sample type |
SRA |
| |
|
| Source name |
cells
|
| Organism |
Saccharomyces cerevisiae |
| Characteristics |
genotype: MAT[alpha] his3 leu2 ura3 lys2 not4::KanMX
culture conditions: YPD exponential growth
library protocol: HT-5PSeq Multiplexed RNA ligation
molecule: total RNA
|
| Treatment protocol |
Strains with a degron were additionally treated for 15 min at 1 mM final.
|
| Growth protocol |
Cells were grown in glucose rich medium (YPD) to exponential phase and for
|
| Extracted molecule |
total RNA |
| Extraction protocol |
Total RNA was prepared by adding acid phenol (200 ul) on the cell pellet together with 200 ul of TES (Tris/EDTA/SDS) (Current Protocols in Molecular Biology (1993)), soluble RNA was prepared by lysing the cell pellet as for polysome profiling (Panasenko and Collart, 2012, doi:10.1111/j.1365-2958.2011.07957.x), and addition of an equal volume of hot acid phenol to the lysis buffer.
HT-5PSeq libraries were generated as reported (Zhang and Pelechano, doi: https://doi.org/10.1101/2020.06.22.165134) with minor modifications. In brief, 15µg total RNA, containing 5% total RNA from Schizosaccharomyces pombe as spike-in, was used. Each sample was spited in two. One part was used for preparing conventional HT-5PSeq libraries and the other part for was random fragmented prior to the preparation of HT-5PSeq libraries (negative control). For random fragmented samples: 7.5ug RNA was fragmentation at 80 ̊C for five minutes in fragmentation buffer (40mM Tris Acetate pH 8.1, 100mM KOAc and 30mM MgOAc). Samples were purified using 1.8x volumes of RNACleanXP beads (Beckman Coulter) following re-phosphorylation of 5’OH sites at 37 ̊C for 60 minutes with 5 Units of T4 Polynucleotide kinase (PNK, NEB). Subsequent, reaction was purified using Phenol:Chloroform: Isoamyl Alcohol (24:25:1), followed by sodium acetate-ethanol precipitation. From this step forward, experimental pipelines for random fragmented and HT-5PSeq library preparation merge. For HT-5PSeq Libraries: 7.5 µg RNA was ligated over night at 16˚C to r5P_RNA_MPX oligo (CrArCrGrArCrGrCrUrCrUrUrCrCrGrArUrCrU rXrXrXrXrXrX rNrNrNrNrNrNrNrN) carrying a sample barcode (rX) and unique molecular identifiers (rN). Ligase was deactivated using 5mM EDTA and heat at 65˚C for 10 minutes (up to X individual barcoded RNA ligations were pooled) and subsequent purified using 1.8x volumes of RNAClean XP beads (Beckman Coulter). Ligated RNA was then reverse transcribed using random hexamer (5Pseq-RT, GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNN, 20µM) and oligo-dT (5Pseq-dT, GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTTTTTTTTTTT at 0.05µM) oligos to prime. After, remaining RNA was degraded using NaOH. Ribosomal RNA was removed using previously descrived rRNA DNA oligo depletion mixes, following a duplex-specific nuclease (DSN, Evrogen) digestion. rRNA depleted cDNA was amplified by PCR (17 cycles) and final product was enriched for fragments with the range of 300-500nt using Ampure XP. Size selected HT-5P Libraries were quantified by fluorescence (Qubit, Thermo Fisher), size estimated using an Agilent Bioanalyzer and sequenced using a NextSeq500 Illumina sequencer (75cycles High output kit). Sequencing files were demultiplexed using bcl2fastq v2.20.0.422 (one mismatch, minimum length 35nt), and adapters were trimmed using cutadapt 2.3. at default settings, allowing one mismatch and minimum read length of 35nt. In addition to standard illumine dual index (i5, i7), the inline sample and UMI barcode was analyzed using Umitools. Reads were mapped to the concatenated genome of S. cervisiae (R64-1-1) and S. pombe (ASM294v2) using STAR. Second read enables to splits reads between oligo-dT or random primer. That information was not used in the current analysis.
|
| |
|
| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina NextSeq 500 |
| |
|
| Data processing |
Library strategy: 5PSeq
Demultiplexing using bcl2fastq v2.20.0.422 at default settings, allowing one mismatch and minimum read length of 35nt
Quality control with FASTQC and MultiQC v1.9 at default parameters
Adapter trimming using cutadapt 2.3 with Python 3.7.2, options: --minimum-length 28; -e 0.2; -o 9; --nextseq-trim 20
Extraction of barcodes and UMI using umitools and options --bc-pattern CCCCCCNNNNNNNN, whitelist.txt file of barcodes was provided and fastq files where spitted according to barcode
Mapping of reads using STAR and options: --alignEndsType Extend5pOfRead1; --outFilterMatchNminOverLread 0.9; --outFilterMultimapNmax 3; --limitBAMsortRAM 100000000000; alignIntronMax 2500
Deduplication using UMIs was done with umitools
Count statistics on species and RNA composition were determined using bedtools intersect (aka intersectBed) Version: v2.29.2, bedtools intersect [OPTIONS] -a <bed/gff/vcf/bam> -b <bed/gff/vcf/bam>
Genome_build: Saccharomyces cerevisiae R64-1-1, Schizosaccharomyces pombe (ASM294v2) and Schizosaccharomyces pombe: GCF_000002945.1_ASM294v2_genomic.fna; GCF_000002945.1_ASM294v2_genomic.gff was created and files were mapped using STAR
Supplementary_files_format_and_content: Bedgraphs of forward (fwd) and reverse (rev) 5P read positions were created using BEDTools genomecov program with options {-bg -strand [+/-] -5} of concatenated Saccharomyces cerevisiae and Schizosaccharomyces pombe genome
|
| |
|
| Submission date |
Mar 04, 2021 |
| Last update date |
Aug 18, 2021 |
| Contact name |
George Allen |
| Organization name |
University of Geneva
|
| Street address |
Rue Michel-Servet 1
|
| City |
Geneve |
| State/province |
Geneve |
| ZIP/Postal code |
1206 |
| Country |
Switzerland |
| |
|
| Platform ID |
GPL19756 |
| Series (1) |
| GSE168290 |
NOT proteins in co-translation decay |
|
| Relations |
| BioSample |
SAMN18143287 |
| SRA |
SRX10240811 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM5135787_not4_tot02_SC_SP_ACTGGT_fwd.bedgraph.gz |
119.7 Kb |
(ftp)(http) |
BEDGRAPH |
| GSM5135787_not4_tot02_SC_SP_ACTGGT_rev.bedgraph.gz |
130.9 Kb |
(ftp)(http) |
BEDGRAPH |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
|
|
|
|
 |
