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Sample GSM6215105

Query DataSets for GSM6215105

Status

Public on Jun 09, 2022

Title

Sample 28_Worm, 48h_6.25×10^-5g/L, rep2

Sample type

SRA

Source name

Whole body

Organism

Caenorhabditis elegans

Characteristics

tissue: Whole body
time: Developed 48 hours from synchronized first larval (L1) stage
genotype: Wild type (N2, Bristol)
treatment: 48h exposure to 6.25×10^-5 g/L HFPO-DA

Treatment protocol

Pre-treatment S Medium solutions with 0, 1.25×10-5, 6.25×10-5, 3.13×10-4, 1.56×10-3, 7.81×10-3, 1.56×10-2, 3.13×10-2, 6.25×10-2, 0.125, 0.25, 0.5, 1, 2, and 4 g/L HFPO-DA were prepared, and 1 ml of each solution was added to a well on 12-well plates. The placement of different concentrations was randomized, and a total of four replicates were prepared for each concentration. Plates with pre-treatment solutions were placed on a plate shaker at 180 rpm overnight. Exposure media with the same concentrations of HFPO-DA and age-synchronized L1 worms were prepared, and the pre-treatment solution in each well was replaced by 1 ml exposure media containing about 1000 worms. Exposure plates were placed on a plate shaker at 180 rpm for 48 hours. At 48 hours after exposures started, worms were pelleted (3000 rpm), washed twice with S Basal to remove residual E. coli HB101, and frozen at -80 ℃.

Growth protocol

The wild-type strain N2 (Bristol) of C. elegans was obtained from Caenorhabditis Genetics Center (CGC) at the University of Minnesota. Worms were maintained at 19 ± 1 ℃ in worm culturing media: S Medium [5.85 g/L NaCl, 1 g/L K2HPO4, 6 g/L KH2PO4, 5 mg/L cholesterol, 10mM potassium citrate (pH = 6.0), 1X N1010 Trace Metal Mix (United States Biological, Salem, Massachusetts, USA), 333 mg/L CaCl2, 361 mg/L MgSO4] with 100 mg/L streptomycin and 10 mg/L nystatin. Escherichia coli (E. coli) HB101 (CGC) were cultured at 37 ± 0.5 ℃ in lysogeny broth (10 g/L tryptone, 5 g/L yeast extract, 5 g/L NaCl, pH = 7.0) with 100 mg/L streptomycin. Pelleted E. coli HB101 were resuspended in S Medium with a final concentration of 25 g/L as the food source for the worms. Worm cultures were maintained as 5-6 mL batches, with each in a cell culture flask (product number: 353109, Falcon®, Corning Inc., Corning, New York, USA), on a plate shaker at 180 rpm.

Extracted molecule

polyA RNA

Extraction protocol

RNA isolation was performed on all 60 samples in the next two days by following TRIzol™ Reagent (catalog number: 15-596-018, Invitrogen™, Waltham, Massachusetts, USA) protocol with slight modifications. Briefly, TRIzol and five 2 mm borosilicate glass beads (product number: CG-1101-01, Chemglass Life Sciences, Vineland, New Jersey, USA) were added to each thawing worm pellet, and a Bead Mill 4 Homogenizer (catalog number: 15-340-164, Fisherbrand™) was used for homogenization. Each RNA sample was resuspended in 40 µL nuclease-free water. The quantity and quality of RNA samples were measured using a NanoDrop 2000 Spectrophotometer (catalog number: ND-2000, Thermo Scientific™, Thermo Fisher Scientific). On the following day, RNA samples were aliquoted and sent to Scripps Research Genomics Core (La Jolla, California, USA) for shallow RNA-sequencing.
cDNA library preparation and shallow RNA-Seq were performed at Scripps Research Genomics Core. Sample quality was assessed using a 2100 Bioanalyzer Instrument (part number: G2939BA, Agilent Technologies, Inc., Santa Clara, California, USA). Library preparation for RNA-Seq was performed via an early access kit, HTP RNA-Seq Library Prep, from iGenomX (now acquired by Twist Bioscience, South San Francisco, California, USA) with slight modifications. Briefly, PCR products 350–800 base pairs long were purified via 2% agarose gel electrophoresis, quantitated using Qubit dsDNA HS assay kit (catalog number: Q33231, Invitrogen™), and analyzed on a 4150 TapeStation System (part number: G2992AA, Agilent Technologies, Inc.).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

NextSeq 2000

Description

pooled around 1000 worms

Data processing

Base call was performed via NextSeq 1000/2000 Control Software Suite v1.4.1
Raw data from shallow RNA sequencing were demultiplexed by Scripps Research Genomics Core using BBTools (version 37.62) (Bushnell et al., 2017).
Unique Molecular Identifiers (UMI) were extracted from the sequencing results using UMI-tools (version 1.1.2) (Smith et al., 2017). Sequencing data quality was assessed in R (R Core Team, 2022. R: A language and environment for statistical computing. R Foundation for Statistical Computing, Vienna, Austria. URL https://www.R-project.org/) via FastQCR (version 0.1.2) (Kassambara, 2019). SortMeRNA (version 4.3.4) was used to filter out rRNA with the sequence database smr_v4.3_sensitive_db.fasta (Kopylova et al., 2012). Trimmomatic (version 0.39) was used to remove the first 16 bases of each read, trim reads with a sliding-window of 4 bases and a minimum Phred score of 30, and remove resulting reads shorter than 18 bases (Bolger et al., 2014). The trimmed reads were aligned to C. elegans genome assembly WBcel235 with genome annotation WS283 via STAR (version 2.7.10a) (Dobin et al., 2013). PCR duplicates were removed using extracted UMI via UMI-tools. Gene counts based on reads mapping were generated through HTSeq (version 2.0.1) (Putri et al., 2022).
Identified genes with non-zero counts were selected from the raw output, and gene expression was normalized via trimmed mean of M-values (TMM) (Robinson & Oshlack, 2010). Average log counts per million (CPM) values were calculated via the aveLogCPM function of edgeR (version 3.36.0) (Robinson et al., 2010) and visualized via ggplot2 (version 3.3.5) (Wickham, 2016). Low count threshold was determined to be 16 counts per million, and low count filtering was performed via the filtered.data function of NOISeq (version 2.38.0) (Tarazona et al., 2015). Because RNA isolation was performed sequentially for the four replicates (batches) of the 15 experimental conditions, potential batch effects were removed via the ARSyNseq function of NOISeq.
Relative to the control condition, pairwise comparison to determine differentially expressed genes for each HFPO-DA exposure concentration was performed through NOISeq, with the probability of differential expression threshold, q, set to 0.99.
Assembly: WBcel235
Supplementary files format and content: worm_normalized.csv; contains the final normalized (as described in the data processing steps) gene counts that entered NOISeq analysis; column titles (Row 1) match the library names, and row titles (Column 1) are gene IDs

Submission date

Jun 06, 2022

Last update date

Jun 09, 2022

Contact name

Goran Bozinovic

E-mail(s)

goran@bozinstitute.org

Phone

19196073155

Organization name

Boz Life Science Research and Teaching Institute