GEO Accession viewer

NCBI > GEO > Accession Display

Not logged in | Login

GEO help: Mouse over screen elements for information.

Sample GSM1380831

Query DataSets for GSM1380831

Status

Public on Jun 04, 2014

Title

CB4856 whole worm (polyA replicate 3)

Sample type

SRA

Source name

whole worm poly(A)-extracted mRNA library

Organism

Caenorhabditis elegans

Characteristics

strain: CB4856
temperature: 24C
rna source: whole worm
library: poly(A)-extracted mRNA library

Treatment protocol

n.a.

Growth protocol

Strains were maintained using previously described methods(Brenner, 1974; Stiernagle, 2006) on OP50 seeded NGM plates at permissive temperatures.

Extracted molecule

total RNA

Extraction protocol

mRNA-Seq: PolyA RNA was fragmented into approximately 250 nt fragments by chemical fragmentation (200 mM tris acetate pH 8.2, 500 mM potassium acetate, 150 mM magnesium acetate) at 94°C for exactly 3.5 min in a thermocycler. Fragmented RNAs were isolated with RNA Clean beads (Beckman Coulter, USA) according to manufacturerâ??s instructions. Fractionation was checked by capillary electophoresis in a RNA Pico 6000 chip using the Bioanalyzer (Agilent Technologies, USA). First strand cDNA synthesis was accomplished using Superscript III Reverse Transcriptase and random hexamers (Invitrogen, USA), followed by second strand synthesis using DNA Polymerase I and RNaseH (Invitrogen USA). Double-stranded DNA was purified with Agencourt AMPure beads XP (Beckman Coulter, USA) and quality was checked by capillary gelelectrophoresis on the Bioanalyzer with the Agilent DNA 1000 kit (Agilent Technologies, USA). dsDNA libraries subsequently processed for sequencing using the Genomic DNA Sample Prep Kit (Illumina, USA) according to the manufacturerâ??s protocol.
Oocytes: Adult wild-type hermaphrodites (N2) were cut using a razorblade in PBS containing 0.5% BSA and 0.02%Tween. Oocytes were picked by mouth pipetting under a stereo microscope (Leica), washed thoroughly in PBS and lyzed in TriZol LS (Invitrogen, USA). Sperm: Sperm was isolated in principle as described previously(L¹Hernault & Roberts, 1995) from males worms obtained from a fog-2(q71) mutant background strain. Males were cut in cold PBS containing protease inhibitors (Sigma-Aldrich, Germany). Sperm was subsequently purified by filtration (3x40µm, 2x10µm, 1x6µm nylon mesh) and washed twice in cold PBS. Sperm was then activated by incubation in PBS containing 200µg/mL Pronase (Sigma-Aldrich, Germany) for 30 minutes at 24°C and suspended in Trizol LS (Invitrogen, USA) for RNA extraction. 1-cell embryos: 1-cell embryos were obtained by fluorescence activated cell sorting (eFACS) as described previously(Stoeckius et al, 2009) in an FACSAriaIII flow cytometer (BD Biosciences, USA). Whole worm: Adult worms were washed from NGM plates with M9-Buffer and subsequently washed at least 3 times with M9-Buffer. RNA was isolated from whole worms by bead homogenization (6000 rpm, 5 seconds) in a Precellys24 (Precellys, USA) in Trizol LS reagent (Invitrogen, USA) according to the manufacturers protocols.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

HW_poly_3
processed data file: data_mRNA_rpkm.txt

Data processing

Sequencing reads for each sample and stage were seperately mapped to the transcriptome using bwa with default parameters. For small RNAs reads were mapped to the genome
For mRNAs reads were aggregated across all isoforms of a given gene.
For mRNAs aggregated reads were converted to reads per million mapped reads per kilobase of transcript length (RPKM).
For small RNAs reads were annotated based on the strand and the annotation (i. e. sense or antisense with regards to a given annotation).
For small RNAs all reads of a given annotation were aggregated and converted into reads per million (RPM)
Genome_build: WS190
Supplementary_files_format_and_content: Tab separated txt-file with gene identifiers and expression values in RPKM for mRNAs and RPM for small RNAs.

Submission date

May 06, 2014

Last update date

May 15, 2019

Contact name

Dominic Grün

E-mail(s)

gruen@ie-freiburg.mpg.de

Phone

+491791073352

Organization name

Max Planck Institute of Immunobiology and Epigenetics