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Status |
Public on Dec 10, 2020 |
Title |
disome-seq_negative_control |
Sample type |
SRA |
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Source name |
Yeast
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Organism |
Saccharomyces cerevisiae |
Characteristics |
treatment: rich medium strain: BY4742
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Treatment protocol |
For experiments using the 3-AT treatment, the strain S288C was cultivated at 30ºC in the SC−His+3-AT medium (synthetic complete medium with histidine dropped-out and 100 mM 3-AT added)
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Growth protocol |
The laboratory strain BY4742 (MATα his3Δ1 leu2Δ0 lys2Δ0 uraΔ0) was cultivated at 30ºC in the rich medium YPD (1% yeast extract, 2% peptone, and 2% dextrose).
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Extracted molecule |
polyA RNA |
Extraction protocol |
Ribosomes were extracted with the polysome lysis buffer (PLB), which contained 200 mM Tris-HCl (pH 8.0), 200 mM KCl, 35 mM MgCl2, 1% (v/v) Triton X-100, 5 mM DTT, and 50 µg/mL cycloheximide. 50000 units (A260) of ribosome dissolved in the PLB buffer were treated with 750 U RNase I (Ambion, AM2294) at 25°C for 2 hours. Our pilot experiments, as well as previous studies, have shown that polysome profiling is not necessary as long as the digestion with RNase I is complete; rather, this step consumes a large amount of ribosome (especially disome) samples. The following-up computational analyses can help to tell if the RNA fragments being collected are largely protected by ribosomes: whether the fragments are restricted to the coding sequences, whether the fragments are enriched in certain sizes, and whether a 3-nt periodicity exists . Therefore, RNA was directly extracted from the enzyme reaction system with hot phenol and was separated on a 17% (w/v) 7 M urea denaturing polyacrylamide gel in a 0.5×Tris-borate-EDTA (TBE) electrophoresis buffer. RNA fragments with the length of 25-30 nts or 50-80 nts were extracted by gel crushing and further incubated with an RNA gel extraction buffer (300 mM NaOAc pH 5.2, 10 mM Tris-HCl pH 8.0, 1 mM EDTA pH 8.0) overnight. The purified extracted RNA fragments for monosome-seq, disomes-seq, and mRNA-seq were subjected to small RNA library construction for Illumina sequencing (Gnomegen, k02420). The 5′-RNA adaptor contained a 3-nt random sequence at the 3′-end to avoid biased ligation. Monosome and disome footprints were sequenced with single-end 50 and paired-end 100 modes on BGISEQ-500 (BGI Group), respectively. The lengths of RNA fragments protected by single ribosomes peaked at 28-nt, consistent with previous studies.
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Library strategy |
OTHER |
Library source |
transcriptomic |
Library selection |
other |
Instrument model |
BGISEQ-500 |
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Description |
disome.neg.peak_28-29nt.txt
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Data processing |
Library strategy: Ribo-Seq (ribosome profiling) Sequenced reads were trimmed for adaptor sequence. The 3-nt random sequence at the 5'-end of each sequencing read was removed(, except for the two disome-seq-3AT libraries). The removed 3-nt sequence was added to the head of each read in the fastq format as the UMI, which can serve to remove PCR duplications generated during Illumina library preparation. The sequence identical to the 3'-sequencing adaptor (AGATCGGAAGAGCACACGTCT for read 1 in both disome-seq libraries and the disome-seq_negative_control; TGGAATTCTCGGGTGCCAAG for all eight monosome-seq and mRNA-seq libraries) was also trimmed in each read using cutadapt V1.16. The trimmed reads were mapped against rRNA with bowtie V1.2.2 with default parameters, and the mapped reads were filtered to avoid rRNA contamination. The rest reads were aligned against SGD R64-1-1 coding sequences with the --no-novel-juncs parameter using Tophat V2.1.1. Genome_build: SGD R64-1-1 Supplementary_files_format_and_content: tab-delimited text files include read number for each Sample
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Submission date |
Dec 10, 2020 |
Last update date |
Dec 30, 2020 |
Contact name |
Yan-Ming Chen |
E-mail(s) |
ymchen@genetics.ac.cn
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Organization name |
Institute of Genetics and Developmental Biology Chinese Academy of Sciences
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Street address |
Beichen West Rd.
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City |
Beijing |
ZIP/Postal code |
100101 |
Country |
China |
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Platform ID |
GPL25927 |
Series (1) |
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Relations |
BioSample |
SAMN17052320 |
SRA |
SRX9670569 |
Supplementary data files not provided |
SRA Run Selector![Help](/coreweb/images/long_help4.gif) |
Raw data are available in SRA |
Processed data are available on Series record |
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