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Sample GSM2465977

Query DataSets for GSM2465977

Status

Public on Feb 13, 2017

Title

YW_18C_2

Sample type

SRA

Source name

3' RNA-Seq (without RNase H treatment)

Organism

Drosophila melanogaster

Characteristics

strain: YW
tissue: head
genotype/variation: wild type
temperature tested: 18C

Growth protocol

BEAF32 experiment: To down regulate beaf-32, we generated flies expressing a UAS-RNAi transgene (BDSC number 35642) under the control of the actin5C-gal4 promoter (Stock #25374, Bloomington Stock Center, Indiana, USA). We utilized actin5C-gal4 flies as control. Flies were reared at 25°C on a standard diet (yeast: 38gr/L, Yellow corn mill: 91gr/L, agar: 10gr/L, molasses: 8.7% v/v, propanoic acid (BioLab): 0.9% v/v, Tegasept solution (Sigma-Aldrich; 300g/L in EtOH (BioLab)): 0.8% v/v)). After eclosion flies were kept in 12-h light:12-h dark cycles at 25°C. Newborn flies were entrained for three days in 12:12 light: dark conditions at different temperatures (18°C, 29°C). Full length RNA-seq and 3’-seq using 100bp: CS fly heads (1 ml) were collected at ZT3 and homogenized in 3 mL of NEB buffer (10 mM HEPES, pH 8.0, 10 mM NaCl, 0.1 mM EGTA, 0.5 mM EDTA, 1 mM DTT, 0.5% Tergitol NP-10, 0.5 mM spermidine, 0.15 mM spermine plus protease inhibitor tablets [Roche])

Extracted molecule

polyA RNA

Extraction protocol

BEAF32 experiment: RNA was extracted from their heads using TRI Reagent, Sigma Aldrich. Full length RNA-seq and 3’-seq using 100bp: RNA was extracted using Trizol (Invitrogen).
BEAF32 experiment: (Dnase treated) RNA was fragmented in the presence of 10XFastAP buffer (ThermoFisher Scientific), at 94°C for 3 minutes, and placed on ice. RNA was then dephosphorylated with FastAP, cleaned (2X SPRI) and ligated to linker1. Library preparation was continued as described for Exo-seq, starting from linker1 3’ ligation. Full length RNA-seq: Full-length RNA libraries were done similarly to the described 'RNAseH- samples', with a difference in the order of the different steps; First, RNA was PolyA+ selected, then fragmented (using Mg-based fragmentation), cleaned (2.5X SPRI cleanup), FastAP treated, cleaned (Silane cleanup) and continued in library preparation (starting at first ligation of 3’ adapter). 3’-seq using 100bp: Library preparation was done as described for RNaseH- samples.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

To down regulate beaf-32, we generated flies expressing a UAS-RNAi transgene (BDSC number 35642) under the control of the actin5C-gal4 promoter (Stock #25374, Bloomington Stock Center, Indiana, USA). We utilized actin5C-gal4 flies as control. RNA was collected at 18C
beaf.32.counts.txt

Data processing

Illumina Casava1.7 software used for basecalling.
Sequenced reads were trimmed for adaptor sequence, and masked for low-complexity or low-quality sequence, then mapped to dm3 whole genome and transcriptome using STAR (for 3’-sequencing using 100bp) or tophat2 (all other samples)
Beaf32 experiment: Read count was calculated using ESAT. Full length RNA-seq and 3’-seq using 100bp were only used for validation of Exo-seq and RNAseH-seq and thus were not processed further.
Genome_build: dm3
Supplementary_files_format_and_content: Expression matrix of the read counts for each genes in wild type and beaf32 knockdown flies

Submission date

Jan 24, 2017

Last update date

May 15, 2019

Contact name

Shaked Afik

E-mail(s)

[email protected]

Organization name

UC Berkeley

Department

Computational Biology Graduate Group