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Sample GSM2861623

Query DataSets for GSM2861623

Status

Public on Oct 10, 2019

Title

N2 rep 1 [day 2]

Sample type

SRA

Source name

whole C. elegans

Organism

Caenorhabditis elegans

Characteristics

genotype: N2
age: day 2

Growth protocol

C. elegans strains were cultured at 20°C under standard conditions as described in Brenner

Extracted molecule

total RNA

Extraction protocol

To analyze RNA from transgenic lines, 100 worms (24 hours post-L4) of each strain (including controls) were placed into 1.5 ml M9 buffer. Worms were washed once in M9, pelleted by centrifugation, resuspended in 200 µl Trizol, vortexed for 2 minutes and flash frozen in liquid nitrogen. Worms were then freeze-cracked by thawing in a 37°C water bath and re-freezing in liquid nitrogen. This was repeated 2 more times. After the final thaw, 100 µl of Trizol was added and the tubes were maintained at room temperature for 5 minutes. RNA was then extracted with 140 µl of chloroform, precipitated with an equal volume of 70% ethanol and transferred to an RNeasy spin column (Qiagen) and purified.
Libraries were prepared using Illumina TruSeq Stranded mRNA sample preparation kits from 500 ng of purified total RNA according to the manufacturer’s protocol. The finished dsDNA libraries were quantified by Qubit fluorometer, Agilent TapeStation 2200, and RT-qPCR using the Kapa Biosystems library quantification kit according to manufacturer’s protocols. Uniquely indexed libraries were pooled in equimolar ratios and sequenced on an Illumina NextSeq 500 with paired-end 75 bp reads by the Dana-Farber Cancer Institute Molecular Biology Core Facilities.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Description

day2_n2_1.1

Data processing

Reads were aligned to the Caenorhabditis elegans WBcel235 genome with Ensembl WBcel235.86 gene models using STAR with options --outFilterMismatchNoverLmax 0.04 --outFilterMismatchNmax 999 --alignSJDBoverhangMin 1 --alignSJoverhangMin 8 --outFilterMultimapNmax 20 --outFilterType BySJout --alignIntronMin 15 --alignIntronMax 1000000 --alignMatesGapMax 1000000. Expression levels of genes were quantified as gene counts using FeatureCounts v1.5.1 with options -C -p -B -s 2 -t exon -g gene_id.
Genome_build: WBcel235
Supplementary_files_format_and_content: Tab-delimited text file indicates gene counts for each sample

Submission date

Nov 20, 2017

Last update date

Oct 10, 2019

Contact name

Derek Drake

Organization name

Harvard Medical School

Department

Genetics

Lab

Yankner