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Sample GSM895827

Query DataSets for GSM895827

Status

Public on Apr 26, 2012

Title

hj37_a

Sample type

SRA

Source name

hj37

Organism

Caenorhabditis elegans

Characteristics

tissue: whole animal
genotype/variation: rde-11 mutant
Stage: 48hrs after L1
tap treatment: No

Growth protocol

Synchronized L1 worms were grown on RNAi plates seeded with HT115 E. coli expressing a sel-1 RNAi trigger as previously described (Pak and Fire 2007). After 48hrs at 20oC, 40000 worms were washed 5 times with 10ml M9, and then homogenized in Tri-reagent. Samples were kept at -80oC.

Extracted molecule

total RNA

Extraction protocol

Purified total RNA were processed with MirVana kit (Invitrogen) for small RNA enrichment. Equal amount of small RNAs (20~30ug) were resolved on 15% TBE-Urea Acrylamide gels. Gels containing small RNAs of 21-24nt were cut out and purified with a small-RNA PAGE Recovery Kit (Zymo Research, R1070). Each small RNA sample was split into two (one for TAP treatment and another untreated) and processed with the ScriptMiner Small RNA-Seq Library Preparation Kit (Epicentre, SMMP1012). Libraries from different samples were indexed with RNA-Seq Barcode Primers (Epicentre, RSBC10948). Two biological samples of each condition (wild type, wild type + TAP, rde-10 mutant, rde-10 mutant + TAP, rde-11 mutant and rde-11 mutant + TAP) were used for library preparation.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

size fractionation

Instrument model

Illumina HiSeq 2000

Description

small RNA

Data processing

The quality of the raw sequence data was analyzed to check if any alteration of the data needed to be done before continuing with downstream analysis. Fastqc was used for a more in-depth look at the quality of the sequence data. All samples were run on the same lane, using TruSeq indexes to distinguish between samples. Overall, sequence quality looks very good for all samples. Clipping is still performed on these samples due to the presence of adapter sequence in the reads as described in the Methods section.
Due to the short read lengths of the small RNA’s, adapter sequence was present at the 5’ end of the reads. Fastx_clipper (v0.0.13) was used to remove this adapter sequence. As the valid portion of the reads are of variable length (the small RNAs range in size), a subset of the adapter sequence was used that looked to be matched in the vast majority of reads. Then, remaining portions of the read smaller than 8 bases long were removed from the resulting clipped sequence file. In this way, reads that contained more of the adapter sequence than what was clipped would still have the rest of the adapter removed, as the length of the remaining adapter was less than 8bp.
Tophat (v1.4.0) was used to perform the alignment of the clipped reads. The UCSC reference genome ce6 was used for alignment and gene annotations. These reference files were downloaded from Illumina’s igenome ftp site. Samtools was used to sort and index the resulting bam files.
Genome_build: ce6

Submission date

Mar 14, 2012

Last update date

May 15, 2019

Contact name

Huan Yang

Organization name

Stowers Institute for Medical Research

Lab

Mak Lab

Street address

1000 East 50th Street