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| Status |
Public on Jun 30, 2018 |
| Title |
JCLIA001D |
| Sample type |
SRA |
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| Source name |
Mid-exponentially grown cells expressing OPT
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| Organism |
Saccharomyces cerevisiae |
| Characteristics |
strains: Engineered D452-2 containing the plasmid pRS316-OPT
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| Extracted molecule |
total RNA |
| Extraction protocol |
(1) Extraction of ribosome-protected mRNA: Frozen cell pellets were lysed by low-frequency cryogenic mixer milling. Lysate was clarified of cell debris and 25 A260 units of extract were treated with 450 U of RNase I (Ambion) for 1 h at room temperature with gentle rotation; digestion was stopped by addition of 120 U of Superase-IN. Ribosomes were pelleted using high-speed centrifugation through a 1 M sucrose cushion. miRNeasy Mini kit (Qiagen) was used to purifiy ribosome-protected mRNA fragments following the manufacturer’s instructions. (2) Extraction of total RNA: Total RNA was extracted using the RiboPure Yeast kit (Ambion, Austin, TX, USA), according to manufacturer's instructions, except cells were disrupted by bead-beating 3 times for 30 sec, with a 30 sec pause between runs.
(1) Ribosome profiling library: Preparation of ribosome profiling libraries followed similar protocols as those in (Ingolia et al., 2012; Ingolia et al., 2009). (2) RNA-seq library: 4 µg of total RNA were used to prepare the multiplexing libraries with barcodes following the standard instructions of the Illumina TruSeq RNA Sample Prep Kit.
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| Library strategy |
OTHER |
| Library source |
transcriptomic |
| Library selection |
other |
| Instrument model |
Illumina Genome Analyzer II |
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| Description |
Ribosome-protected mRNA
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| Data processing |
Illumina Casava_v1.8.0 software used for basecalling. Sequenced reads were trimmed for adaptor sequence.
For footprint analysis, trimmed sequencing reads without the linker were aligned to the S. cerevisiae ribosomal sequences using Bowtie (Langmead et al., 2009). These reads were removed, and the non-rRNA reads were then mapped to the S. cerevisiae genome using Tophat (Trapnell et al., 2009). The sequence for the pRS316-NC/OPT plasmids were manually annotated and added to separate genome fasta files created for each condition and designated as new chromosomes. Only uniquely aligned reads were used for subsequent analyses. Most of the reads were between 27-32 nt long and these reads were mapped onto their respective coding sequences as described previously (Ingolia et al., 2012; Ingolia et al., 2009).
For RNA-seq data, sequence reads were assembled and analyzed in CLC Genomics Workbench 6.5 (CLC Bio, Aarhus, Denmark). The genome for S. cerevisiae S288C (version R64.2.1), the parent strain of D452-2 , was downloaded from Refseq at the NCBI (http://www.ncbi.nlm.nih.gov/refseq/); the mitochondrial genome was included. The sequence for pRS316-NC and -OPT were manually annotated and added to this file. Expression values were normalized by calculating the reads per kb of mRNA per million mapped reads (RPKM), and normalized further by using the option of “By totals”.
Genome_build: Saccharomyces cerevisiae S288C (version R64.2.1)
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| Submission date |
Nov 13, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Ligia Acosta-Sampson |
| E-mail(s) |
lacostasampson@berkeley.edu
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| Phone |
510-666-2559
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| Organization name |
UC Berkeley
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| Department |
MCB/EBI
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| Lab |
Jamie H.D. Cate
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| Street address |
2151 Berkeley Way, Room 320, MC 5230
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| City |
Berkeley |
| State/province |
CA |
| ZIP/Postal code |
94720 |
| Country |
USA |
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| Platform ID |
GPL9377 |
| Series (1) |
| GSE75004 |
Translation elongation mediated quality control of integral membrane protein synthesis |
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| Relations |
| BioSample |
SAMN04267391 |
| SRA |
SRX1431874 |
| Supplementary file |
Size |
Download |
File type/resource |
| GSM1940308_JCLIA001D_CGACGT_unique_fwd.wig.gz |
1.7 Mb |
(ftp)(http) |
WIG |
| GSM1940308_JCLIA001D_CGACGT_unique_rev.wig.gz |
1.7 Mb |
(ftp)(http) |
WIG |
SRA Run Selector |
| Raw data are available in SRA |
| Processed data provided as supplementary file |
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