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Sample GSM3056291

Query DataSets for GSM3056291

Status

Public on Jul 13, 2018

Title

fog2_516

Sample type

SRA

Source name

L1 larvae

Organism

Caenorhabditis elegans

Characteristics

developmental stage: L1 larvae
strain: fog-2
l1 arrest density: 5 egg/ul

Treatment protocol

For L1s arrested at different densities eggs were reconsititued at 1, 5, 20, or 100 eggs/ul of M9 and hatched/arrested overnight.

Growth protocol

Animals were passaged by bleaching gravid worms and directly plating 125,000 eggs mixed with E. coli OP50 (washed and resuspended in M9 buffer to 0.33 mg/mL), therefore worms never experience starvation under these conditions. Strains were maintained on 150 x 15 mm petri dishes that contained standard nematode growth media (NGM) with modification [10g agarose + 7g agar instead of 17g agar]. Gravid animals were washed from plates with M9 buffer, collected in conical tubes and centrifuged at 2000 x g for 30 seconds. Next, the supernatant was removed and worms were treated with 10 mL of bleach solution [41:6:3 ddH2O, sodium hypochlorite (Fisher Chemical, SS290-1), 5M KOH, and kept on a rocking platform. Worms were spun down after 4 minutes and the supernatant was replaced with fresh bleach. This procedure was repeated one more time and the suspension was visually checked to ensure all worm bodies were dissolved. The suspension was centrifuged at 2000 x g for 30 seconds, bleach solution was removed, and embryos were washed with M9 buffer supplemented with PEG 3350 (0.5%, w/v) three times before use. In this study, M9 buffer was always supplemented with PEG 3350 (0.5%, w/v) unless noted otherwise. The density of embryos in M9 were determined by manual counting.

Extracted molecule

total RNA

Extraction protocol

Single L1 animals were placed in 5 ul of a typical worm lysis buffer (40mM Tris pH7.5, 10mM EDTA, 200mM NaCl, 0.5% SDS, and 0.4ug/ul Proteinase K), incubated at 55° for 10 minutes, and then frozen at -20°C until use for RNA-sequencing.
single-L1 RNA-seq/Nextera kit

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NextSeq 500

Data processing

Reads were aligned to ce11 RefSeq using Bowtie software in a sequencial manner first to rRNAs to eliminate any reads coming from them and then aligned to the genome using RSEM. During RSEM duplicated reads were removed.
Genome_build: ce11 ws245
Supplementary_files_format_and_content: excel sheet with normalized gene counts

Submission date

Mar 19, 2018

Last update date

Jul 13, 2018

Contact name

Colin Conine

E-mail(s)

[email protected]

Organization name

UPenn Perelman SOM and CHOP

Department

Genetics and Neonatology