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Sample GSM1660704

Query DataSets for GSM1660704

Status

Public on Oct 07, 2018

Title

gld-2 rep2

Sample type

SRA

Source name

gld-2 rep2

Organism

Caenorhabditis elegans

Characteristics

strain: gld-2(q497)
developmental stage: young adult
tissue: whole worm

Treatment protocol

To induce RNAi, C. elegans N2 (wildtype) worms were grown on 15 cm plates with Nematode Growth Medium spotted with concentrated E. coli HT115(DE3) expressing dsRNA of interest.

Growth protocol

C. elegans N2 (wildtype) worms were grown on 15 cm plates with Nematode Growth Medium spotted with concetrated E. coli OP50.

Extracted molecule

total RNA

Extraction protocol

Total RNA was extracted from snap frozen cell pellets Trizol extraction. 1 microgram of this was input into the library preperation.
Library construction was by the PAT-seq approach. Breifly this involves a modified the ePAT approach to tagging adenylated RNA to generate libraries suitable for deep sequencing. Briefly, adenylated RNA is sequence specifically extended by dNTPs using Klenow polymerase and an annealed DNA anchor oligonucleotide. This takes advantage of the native function of DNA polymerase to extend an RNA primer from a DNA template in second strand synthesis. Importantly, any unwanted priming to internal poly(A)-tracts in RNA is avoided by a requirement for 3’ extension in subsequent fragment selection and reverse transcription. No ribosome depletion is necessary. Here, the anchor sequence was compatible with the Illumina index primers and included a 5’ biotin moiety to facilitate handling. In a second step, the 3’ tagged RNA was subject to limited fragmentation by RNase T1. This cleaves RNA after G-residues and ensures that cleavage is only possible within the body of the RNA, not the poly(A)-tract or the DNA sequence of the extended tag. The fragmented RNA was 5’ phosphorylated to allow RNA Ligase 2 mediated ligation of an Illumina compatible splinted-linker to the RNA fragments. Reverse transcription was primed from the anchor sequence. Note: All manipulations after limited fragmentation were performed in association with streptavidin magnetic beads. The cDNA PAT-seq libraries was eluted from beads, size-selected by Urea PAGE and amplified with primers that introduce the features for directional Illumina sequencing and indexing. In samples analysed here, the window of selection was between 120-300 bases. This size range was selected to allow for ≥ 25 bases of 3’UTR sequence to map reads to the genome, the an average yeast poly(A)-tail of ~25 bases (maximum ~90 bases), the majority of reads would contain heterogeneous 5’ sequence of sufficient length to map uniquely to the yeast genome. Note: all reads run in 5’ to 3’ direction from unique sequence into a variable length of poly(A) homopolymers. This means that color balance is preserved and that any low fidelity within the homopolymers is limited to the end of the read.
9pM of libraries per lane using Illumina c-bot. Illumina protocol 15006165 Rev J, July 2012
1 x 150bp sequencing using Illumina protocol 15035788 Rev A, Oct 2012

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina MiSeq

Description

Run 1, Illumina index 15
Protocol: PAT-Seq

Data processing

Processing is automated using the Tail Tools pipeline (https://github.com/Victorian-Bioinformatics-Consortium/tail-tools), and proceeds as follows:
Reads are stripped of trailing poly(A) and adaptor sequence (allowing for up to 1 in 5 sequencing errors).
Reads are aligned using Bowtie2 (v2.2.4, http://bowtie-bio.sourceforge.net/bowtie2/index.shtml). Multi-mapping reads are assigned a location at random from the best alignments.
Alignments are extended where the stripped poly(A) sequence matches the reference genome. The remaining, untemplated, poly(A) length is taken as the poly(A) tail length for that read. A read is called as having a poly(A) tail if this length is at least 4 As.
Reads aligning to genes are counted. Transcript locations were downloaded from the Refseq track of the UCSC genome browser. Overlapping transcripts (mostly isoforms) were merged. As 3'UTR annotations are not always accurate, reads up to 1000 bases down-strand of a gene are counted towards that gene. Where a read might be assigned to multiple genes, it is assigned to the gene with annotated 3' end nearest to the alignment's 3' end. Total reads per gene, reads with a poly(A) tail per gene, and average poly(A) tail length are tabulated.
Genome_build: ce10
Supplementary_files_format_and_content: count.csv contains counts of reads aligning to each gene in each sample. tail_count.csv contains counts of reads with a poly(A) tail aligning to each gene in each sample. This is used when detecting differential tail length as the more reads there are, the higher the accuracy of the average in tail.csv. tail.csv contains the average poly(A) tail length observed in reads with a poly(A) tail for each gene in each sample. These files are sufficient for differential expression and differential tail length analysis with the statistics package "Fitnoise". gene.csv lists the genomic locations of the genes that were used. Locations are 1-based.

Submission date

Apr 17, 2015

Last update date

May 15, 2019

Contact name

Traude Beilharz

E-mail(s)

traude.beilharz@monash.edu

Organization name

Monash University

Department

Biomedicine Discovery Institute