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Sample GSM1399294

Query DataSets for GSM1399294

Status

Public on May 25, 2015

Title

pos-1 (RNAi) rep2

Sample type

SRA

Source name

N2 (pos-1 RNAi)

Organism

Caenorhabditis elegans

Characteristics

strain backround: N2 (pos-1 RNAi)
developmental stage: Early embryos (> 90% zygote to 24 cell stage)

Treatment protocol

To induce RNAi, C. elegans N2 (wildtype) worms were grown on 15 cm plates with Agar Nematode Growth Medium spotted with concetrated E. coli HT115(DE3) expressing dsRNA of interest, cultured in terrific broth and induced with IPTG for 6 hours in the culturing liquid media

Growth protocol

C. elegans N2 (wildtype) worms were grown on 15 cm plates with Agar 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.
Cluster Generation: 9pM of libraries per lane using Illumina c-bot. Illumina protocol 15006165 Rev J, July 2012
Sequencing chemistry: 1 x 150bp sequencing using Illumina protocol 15035788 Rev A, Oct 2012

Library strategy

OTHER

Library source

transcriptomic

Library selection

other

Instrument model

Illumina HiSeq 1500

Description

Illumina index 8

Data processing

Library strategy: PAT-seq
All analysis was carried out using Tail Tools version 0.29 available from http://www.vicbioinformatics.com/software.tail-tools.shtml
Reads were first clipped of poly(A) and adaptor sequence: The read was searched for a run of "A"s extending to the end of the read, or a run of "A"s extending into the adaptor sequence. An error rate of one base in five was allowed, and read bases with quality below 10 were ignored.
Clipped reads were then aligned to the reference genome using Bowtie version 2.2.2. Where a read had several equal best alignments, one was chosen at random.
Alignments which were followed by As in the reference genome were extended to cover these As if they were also seen in the original read. We refer to the number of non-templated As in a read as its tail length. Reads with tail length of at least 4 are referred to as poly(A) reads below.
Reads were assigned to genes if their alignment overlapped the region from the 5' end of the gene to 1000 bases 3' of the 3' end of the gene. If this would assign a read to multiple genes, the gene minimizing the distance between the 3' end of the alignment and the 3' end of the gene was chosen.
From this a count of reads per gene is obtained. Where a gene has at least 10 poly(A) reads, the average tail length of poly(A) reads is also calculated for that gene. This statistic provides information about the poly(A) tail length of transcripts. It is expected to be an underestimate as the whole poly(A) tail is not always read, except in the case of poly(A) tails shorter than 12 bases, in which case it may be an overestimate.
Polyadenylation sites were called where the 3' end of the alignments of at least 50 poly(A) reads occurred within 10 bases of each other. Where multiple candidate sites existed within 50 bases of each other, only the site with the greatest number of poly(A) reads was called.
Reads were assigned to polyadenylation sites if their alignmnet overlapped a region from 100 bases 5' of the site to the site itself. Again, if a read could be assigned to multiple polyadenylation sites the site minimizing the distance to the 3' end of the alignment was chosen.
As with genes, read counts and average tail lengths were calculated for each called polyadenylation site.
Genome_build: ce10
Supplementary_files_format_and_content: genes.gff contains the genome region used for each gene.
Supplementary_files_format_and_content: genewise-counts.csv contains counts of reads aligning to these regions or up to 1000 bases downstrand.
Supplementary_files_format_and_content: genewise-tails.csv contains average poly(A) read tail lengths for each gene.
Supplementary_files_format_and_content: peaks.gff contains the called polyadenylation sites. The polyadenylation site is the 3' end of each of these features.
Supplementary_files_format_and_content: peakwise-counts.csv contains counts of reads aligning to each polyadenylation site.
Supplementary_files_format_and_content: peakwise-tails.csv contains average poly(A) real tail lengths for each polyadenylation site.

Submission date

May 27, 2014

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