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Sample GSM5272995

Query DataSets for GSM5272995

Status

Public on May 01, 2021

Title

WT C. elegans control rep.2 [Control_rep2_04_12]

Sample type

SRA

Source name

WT C. elegans control

Organism

Caenorhabditis elegans

Characteristics

strain background: N2
age: 5 days old nematodes
treatment: none (control)

Treatment protocol

50µM CGP37157 seeded plates, incubated at 20ºC

Growth protocol

For worm synchronization, eggs were placed overnight in NGM plates without OP50 to cause L1 larvae arrest. After 12 hours, L1 larvae were transferred to NGM plates seeded with OP50 and let develop at 20ºC. As soon as they reach the young adult stage, worms were transferred to control and treatment (50µM CGP37157) seeded plates, incubated at 20ºC and transferred every day to new plates. At day 5 of treatment, total RNA extraction was performed.

Extracted molecule

total RNA

Extraction protocol

control and treated worms were collected with distilled H2O, transferred to a Falcon tube and washed three times to eliminate eggs or larvae left in the plates. Then they were transferred to an eppendorf tube, the rest of the supernatant was carefully removed and three freezing-unfreezing cycles in liquid nitrogen were performed. Then, 10-20 silica beads and 500 µl of TRizolTM were added to each frozen sample, samples were homogenized using a beads homogenizer for 1 minute and incubated in ice for 5 more minutes. Then 100µl of chloroform were added to each sample, incubated for 3 minutes and centrifuged at 4ºC, 15 minutes at 15.000 rpm. The upper phase was then transferred to another eppendorf tube and finally, a solution 1:1 (v/v) of the sample and Ethanol 70% was prepared. For RNA purification, the QIAGEN RNEasy Mini Kit was used.
1 microgram RNA was prepared using TruSeq Stranded mRNA kit, following Illumina instructions. RNA quality and quantity was assesed using a Fragment Analyzer (AATI) and Qubit (ThermoFisher). Libraries run on a HiSeq1500 Illumina in 100 cicles paired-end reads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 1500

Data processing

Mapping vs reference genome (HISAT2)
Analysis of differential expression (multi QC)
Reads count (FeatureCounts)
Sttistical analysis (DESeq2, EdgeR, SARTools)
Supplementary_files_format_and_content: DESeq/EdgeR expression analysis

Submission date

Apr 30, 2021

Last update date

May 02, 2021

Contact name

Javier Alvarez

E-mail(s)

jalvarez@ibgm.uva.es

Phone

+34 983184844

Organization name

IBGM, CSIC / Univerity of Valladolid