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Sample GSM6808641

Query DataSets for GSM6808641

Status

Public on Dec 12, 2022

Title

Unc-1 mutant, biological rep 1

Sample type

SRA

Source name

whole organism

Organism

Caenorhabditis elegans

Characteristics

tissue: whole organism
cell line: RVM10
cell type: whole organism
genotype: 40Q::YFP; unc-1(vlt10)
treatment: 20 ºC, young adult

Treatment protocol

We sequenced 4 different genotypes, with 6 biological replicates per genotype.

Growth protocol

Synchronized L1 animals were cultured in NGM plates at 20 °C until they reached the young adult stage.

Extracted molecule

total RNA

Extraction protocol

Yound adults were collected in M9 buffer, frozen, thawed and mechanically lysed with 200 mg of glass beads (Sigma-Aldrich-Merck) for 30 sec using a Fastprep shaker apparatus (Thermofisher Scientific, FP120 model) . tRNA was extracted using NZY total RNA isolation kit, Nzytech. Supernatant was recovered from lysate samples by centrifuging extracts at maximum speed for 1 min. The purified RNA was treated with DNAse, quantified and sent for sequencing by Novogene (Cambridge, UK).
Library preparation was performed at Novogene facilities (Cambridge, UK). mRNA was enriched using oligo(dT) beads and then fragmented randomly in fragmentation buffer, followed by cDNA synthesis using random hexamers and reverse transcriptase. After first-strand synthesis, a custom second-strand synthesis buffer (Illumina) was added with dNTPs, RNase H and Escherichia coli polymerase I to generate the second strand by nick- translation. To build the final cDNA library, a round of purification, terminal repair, A-tailing, ligation of sequencing adapters, size selection and PCR enrichment were performed. Library concentration was first quantified using a Qubit 2.0 fluorometer (Life Technologies). Insert size was checked on an Agilent 2100 and quantified using quantitative PCR (Q-PCR).

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina NovaSeq 6000

Description

R1_unc_1
genes.readcount.annot.xls
genes.FPKM.annot.xls
unc_1_vs_WT.genes.DEA.xls
unc_1_vs_nhr_1.genes.DEA.xls
U_N_vs_unc_1.genes.DEA.xls

Data processing

Data filtering: Raw reads are filtered to remove reads with adapter contamination or reads with low quality. Only clean reads were used in the downstream analyses. The filtering process is as follows: (1) Discard reads with adaptor contamination. (2) Discard reads when uncertain nucleotides constitute more than 10% of either read (N > 10%). (3) Discard reads when low quality nucleotides (base quality less than 20) constitute more than 50% of the read. RNA-seq Adapter sequences (Oligonucleotide sequences of adapters from TruSeqTM RNA and DNA Sample Prep Kits): RNA 5' Adapter (RA5): 5'-AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACGCTCTTCCGATCT-3' RNA 3' Adapter (RA3): 5'-GATCGGAAGAGCACACGTCTGAACTCCAGTCAC(6-nucleotide index)ATCTCGTATGCCGTCTTCTGCTTG-3'
Mapping: HISAT2 v2.0.5 with default parameters
Quantification: HTSeq v0.6.1 with -m union option.
Differential expression analysis: DESeq2 v1.24.0
Assembly: WBCel235
Supplementary files format and content: .bam file for each sample (24 total)
Supplementary files format and content: .xls file with raw counts for each sample (1 column per sample)
Supplementary files format and content: .xls file with FPKM counts for each sample (1 column per sample)
Supplementary files format and content: .xls file from differential expression analysis for each pair of experimental groups (10 files total)

Submission date

Dec 11, 2022

Last update date

Dec 12, 2022

Contact name

Carlos Mora-Martínez

E-mail(s)

carmoma9@gmail.com

Organization name

Universidad Internacional de Valencia

Street address

Emilio Lluch 4Dup 16