U.S. flag

An official website of the United States government

Format

Send to:

Choose Destination

SRX878013: GSM1611597: frac_28-35nt; Caenorhabditis elegans; OTHER
2 ILLUMINA (Illumina HiSeq 2000) runs: 66.6M spots, 3.3G bases, 1.5Gb downloads

Submitted by: Gene Expression Omnibus (GEO)
Study: Transcriptome-wide measurement of ribosomal occupancy by ribosome profiling
show Abstracthide Abstract
Experiments for optimization of the ribosome profiling protocol using C. elegans lysates. We report that optimizing the cutting accuracy to a narrow window of 28-30 nucleotides when PAGE-purifying the ribosome-protected fragments (RPFs) significantly improves the quality of the RPF library. In addition, we find that purifying monosomes by sucrose gradient fractionation clearly removed more contaminating ribosomal RNA from the samples compared to the purification by gel filtration columns. Overall design: Experiment 1 (samples 1-4): Comparison between different methods to isolate monosomes and between different library preparations. Experiment 2 (samples 5-8): Optimization of the cutting accuracy to PAGE purify ribosome-protected fragments.
Sample: frac_28-35nt
SAMN03349117 • SRS845969 • All experiments • All runs
Library:
Instrument: Illumina HiSeq 2000
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: Worms were lysed, monosomes purified and RNA was isolated using Tri-reagent (MRC). The RNA from the monosomal fraction was separated using a 15% TBE-Urea Gel (Invitrogen) and the region around 28-30 nucleotides (or the region indicated) excised to isolate Ribosome protected fragments (RPFs). The gel piece was forced through a pierced small tube inside an eppendorf tube by centrifugation and RNA from the gel debris was eluted by overnight incubation in 600 µl cracking buffer (20 mM Tris-HCl (pH 7.9), 1 mM EDTA, 400 mM NH4Acetate, 0.5 % SDS). RNA was precipitated with isopropanol at -80 °C for at least 4 hours (isopropanol precipitation). RPFs were 3’ dephosphorylated with 10 Units of T4 polynucleotide kinase (NEB) in T4 PNK buffer with 40 Units of RNasin for 1 hour at 37 °C. Following isopropanol precipitation, the RNA samples were ligated to 3’ adapters according to the Illumina® TruSeq™ Small RNA Sample Preparation protocol and using the reagents of the kit, then again precipitated with isopropanol. Ligation products were 5’ phosphorylated for 30 minutes at 37 °C with 15 Units of T4 polynucleotide kinase (NEB) in T4 PNK buffer, 1 mM ATP and 40 Units of RNasin. Following heat-inactivation of the enzyme for 10 minutes at 70 °C, the RNA was precipitated by isopropanol. Ligation to 5’ adapters, reverse transcription, PCR amplification with barcoded primers and gel-purification of the PCR products were performed using the Illumina® TruSeq™ Small RNA Sample Prep kit. The following barcodes were used: RPIX 2, 4, 6, 7, 12
Experiment attributes:
GEO Accession: GSM1611597
Links:
External link:
Runs: 2 runs, 66.6M spots, 3.3G bases, 1.5Gb
Run# of Spots# of BasesSizePublished
SRR180434950,000,0002.5G1.2Gb2018-02-12
SRR180435016,596,977829.8M397.2Mb2018-02-12

ID:
1259601

Supplemental Content

Search details

See more...

Recent activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...