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Sample GSM3673631

Query DataSets for GSM3673631

Status

Public on Jan 21, 2020

Title

10 day T3 leg

Sample type

SRA

Source name

embryonic T3 legs

Organism

Schistocerca gregaria

Characteristics

Stage: embryo
age: 10 days
tissue: T3 leg

Growth protocol

Eggs were incubated at 30 degrees Celsius.

Extracted molecule

total RNA

Extraction protocol

Legs and pleuropodia were dissected from embryos of Schistocerca gregaria at indicated age (day) in phosphate buffer saline (PBS), then frozen in liquid nitrogen. Total RNA was extracted using Trizol, quantified using Nanodrop and sent to BGI (Hong Kong) (samples from stages 4, 5, 6, 7, 8, 10, 11, 12 and 13 days) or delivered to EASIH (Cambridge) (sample 8-9 days) for library preparation and sequencing.
Libraries were prepared according to Illumina protocol. The total RNA was enriched in mRNA by using the oligo(dT) magnetic beads.

Library strategy

RNA-Seq

Library source

transcriptomic

Library selection

cDNA

Instrument model

Illumina HiSeq 2000

Description

Sgre_pleuropodia_CountMatrix_filtered.xlsx
Sgre_secreting_pleuropodia_DEGs.xlsx
LEG_10d

Data processing

The quality of the sequenced reads was assessed using the FastQC software. All samples consistently showed a Per base sequence quality of >30. Reads were mapped to the reference transcriptome with Bowtie2 (version 2.2.5) using default parameter and the –local alignment mode (Langmead et al., 2009). The trimmed pairs of reads were concatenated for each stage and treated as single reads.
Sample 8-9 days: Prior to mapping, the 75bp PE reads were trimmed to 50 bp, using Trimmomatic in the paired-end mode (version 0.36) using the CROP function (CROP:50).
The R package HTSFilter (Rau et al., 2013) was used with default parameters to filter constantly low expressed genes
Differential gene expression analysis on active pleuropodia ("highly secreting"), samples 10 days, 11 days, 12 days: The differential expression analysis was performed with the NOISeq R package (2.22.1; Tarazona et al., 2011). Reads were first normalized using the RPKM method (Mortazavi et al., 2008). Samples from day 10, 11 and 12 were treated as replicate and analyzed using the NOISeq-real algorithm with the following parameters: k=0.5, norm="n", factor="type", nss=0, lc=1, replicates = "technical".
Genome_build: Reads were mapped to a reference embryonic transcriptome prepared by us. This Transcriptome Shotgun Assembly project has been deposited at DDBJ/EMBL/GenBank under the accession GHHP00000000. The version used in the study is the first version, GHHP01000000.
Supplementary_files_format_and_content: Excel (xlsx) files. File "Sgre_pleuropodia_CountMatrix_filtered.xlsx" contains a count matrix for each sample after filtering using the HTSFilter (see "data processing"). File "Sgre_secreting_pleuropodia_DEGs.xlsx" contains output from differential expression analysis performed using NOISeq on samples from highly secreting pleuropodia and similarly aged legs. This includes "RPKM" that measures the transcript abundance.

Submission date

Mar 15, 2019

Last update date

Jan 21, 2020

Contact name

Barbora Konopova

E-mail(s)

barbora.konopova@entu.cas.cz

Organization name

Biology Centre CAS

Department

Institute of Entomology