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| Status |
Public on Jan 11, 2016 |
| Title |
poly6_rep2 |
| Sample type |
SRA |
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| Source name |
HEK293T cell culture
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| Organism |
Homo sapiens |
| Characteristics |
cell line: HEK293T
fraction: Six ribosomes
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| Treatment protocol |
Media was aspirated and replaced by PBS + 100 ug/ml cycloheximide and incubated at 37 °C for ten minutes. The dish was then placed on ice, media aspirated, and replaced by ice cold PBS + 100 ug/ml cycloheximide. Cells were scraped, pelleted at 16,000g for 30 seconds, and resuspended in three pellet-volumes ice cold hypotonic lysis buffer (10 mM HEPES pH 7.9, 1.5 mM MgCl2, 10 mM KCl, 0.5 mM DTT, 1% Triton X-100 and 100 ug/ml cycloheximide) (Folco et al., 2012). After ten minutes, cells were lysed on ice by ten strokes through a 26-gauge needle and nuclei were pelleted at 1,500g for 5 minutes. Lysate from ~15 million cells was layered on top of triplicate 10-50% (w/v) sucrose gradients (20 mM HEPES pH 7.6, 100 mM KCl, 5 mM MgCl2, 1 mM DTT and 100 ug/ml cycloheximide) made using a BioComp gradient master. Gradients were centrifuged for 2 hours at 36,000 RPM in a SW-41 rotor, punctured, and manually peak fractionated using real-time A260 monitoring with a Brandel gradient fractionator and ISCO UA-6 detector.
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| Growth protocol |
HEK293T cells were grown to ~70% confluency in DMEM + 10% FBS and were actively growing when harvested
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| Extracted molecule |
total RNA |
| Extraction protocol |
RNA was extracted from pooled technical triplicate sucrose gradient fractions by ethanol precipitation followed by acid phenol:chloroform extraction. Direct phenol:chloroform extraction was precluded by phase inversion in high sucrose fractions. RNA was then DNase treated, subjected to a second acid phenol:chloroform extraction, and ethanol precipitated. Cytoplasmic RNA was extracted using TRIzol extraction (Life Technologies) and ethanol precipitation. Total RNA integrity was verified using a BioAnalyzer (Agilent).
Ribosomal RNA was then depleted using Ribo-Zero (Illumina) and biological duplicate sequencing libraries were generated using the TruSeq RNA Sample Prep v2 kit (Illumina) without the poly-A selection steps. An equal mass of rRNA-depleted RNA was used as input to each individual library preparation.
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| Library strategy |
RNA-Seq |
| Library source |
transcriptomic |
| Library selection |
cDNA |
| Instrument model |
Illumina HiSeq 2500 |
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| Description |
rRNA-depleted
Polysomal RNA
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| Data processing |
Basecalls performed using CASAVA version 1.8
Adapters were trimmed using Cutadapt v1.5, reads aligning to the repeatmasker or Illumina iGenomes abundant sequences using Bowtie2 v2.2.4 were discarded. Unaligned reads were then aligned to the Ensembl release 75 transcriptome using Tophat v2.0.13.
Transcript isoform level abundances were calculated using Cuffquant v2.2.1 and normalized between samples with Cuffnorm v2.2.1.
RPKM from Cuffnorm was converted to TPM as in Wagner, Kin & Lynch 2012.
Genome_build: GRCh37
Supplementary_files_format_and_content: CSV files include TPM (transcripts per million) for all Ensembl transcripts and genes in release 75.
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| Submission date |
May 29, 2015 |
| Last update date |
May 15, 2019 |
| Contact name |
Stephen N. Floor |
| E-mail(s) |
stephen.floor@ucsf.edu
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| Organization name |
University of California, San Francisco
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| Department |
Cell and Tissue Biology
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| Lab |
Floor Lab
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| Street address |
513 Parnassus Ave, HSW 740
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| City |
San Francisco |
| State/province |
CA |
| ZIP/Postal code |
94143 |
| Country |
USA |
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| Platform ID |
GPL16791 |
| Series (1) |
| GSE69352 |
Tunable protein synthesis by transcript isoforms in human cells (Transcript Isoforms in Polysomes sequencing: TrIP-seq) |
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| Relations |
| BioSample |
SAMN03743577 |
| SRA |
SRX1042230 |
| Supplementary data files not provided |
SRA Run Selector |
| Processed data are available on Series record |
| Raw data are available in SRA |
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