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Sample GSM1611591

Query DataSets for GSM1611591

Status

Public on Feb 12, 2018

Title

01_N2_893_SGop

Sample type

SRA

Source name

Whole-worm

Organism

Caenorhabditis elegans

Characteristics

strain: N2
age: young adult

Treatment protocol

Worms were washed of the plates and subsequently washed three additional times with M9 before extraction of RNA

Growth protocol

Egg-prepped C. elegans eggs were allowed to hatch overnight, hatched L1s were plated on peptone rich 2% NGM plates with NA22 for the desired period of time before harvesting

Extracted molecule

total RNA

Extraction 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

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 2000

Description

Monosomes were purified using sucrose gradient fractionation and the library was prepared using the library construction protocol described below.

Data processing

Library strategy: ribosome profiling
Basecalls performed using RTA v1.17.21.3(sample 5-7) or v1.13.48(other samples). Data converted to FastQ file by bcl2fastq v1.8.4(sample 1-4) or v1.8.3(sample 5-8).
Sequencing adaptors were trimmed using Flexbar v2.5.
rRNA reads were removed using Bowtie2 v2.2.4.
Reads were mapped to reference genome using STAR v2.4. Only unique-mapped reads were kept.
Counting number of reads aligned into CDS regions using costom Julia program.
Calculating RPKM value based on read numbers in CDS regions by 1e+9 * #read / ( #(total CDS reads) * sum_of_CDS_lengths )
Genome_build: WS230
Supplementary_files_format_and_content: tab-delimited text files include CDS-RPKM values for each sample (column) and each coding gene (row).

Submission date

Feb 14, 2015

Last update date

May 15, 2019

Contact name

Jieyi Xiong

Organization name

VIB-KULeuven Center for Cancer Biology

Street address

Herestraat 49, box 912

City

Leuven