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Sample GSM1940314 Query DataSets for GSM1940314
Status Public on Jun 30, 2018
Title JCLIA002D
Sample type SRA
 
Source name Mid-exponentially grown cells expressing OPT
Organism Saccharomyces cerevisiae
Characteristics strains: Engineered D452-2 containing the plasmid pRS316-OPT
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.
 
Library strategy RNA-Seq
Library source transcriptomic
Library selection cDNA
Instrument model Illumina Genome Analyzer II
 
Description The_transcriptome_profiles-lacostasampson.xlsx
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)
 
Submission date Nov 13, 2015
Last update date May 15, 2019
Contact name Ligia Acosta-Sampson
E-mail(s) lacostasampson@berkeley.edu
Phone 510-666-2559
Organization name UC Berkeley
Department MCB/EBI
Lab Jamie H.D. Cate
Street address 2151 Berkeley Way, Room 320, MC 5230
City Berkeley
State/province CA
ZIP/Postal code 94720
Country USA
 
Platform ID GPL9377
Series (1)
GSE75004 Translation elongation mediated quality control of integral membrane protein synthesis
Relations
BioSample SAMN04267397
SRA SRX1431880

Supplementary data files not provided
SRA Run SelectorHelp
Raw data are available in SRA
Processed data are available on Series record

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