title: GSM4661641: S10_M_2; Plutella xylostella; RNA-Seq
library_construction_protocol: To determine P. xylostella gene expression responses to cold stress in male and female larvae, total RNA samples were collected from mid-fourth instar larvae treated with 10C and 25C (control) regime. This included 12 samples includes three biological replicates for each temperature and sex. Each biological replicate consists eight P. xylostella larvae. Total RNA of whole bodies of samples in each replicate was extracted using Qiazol lysis reagent according to manufacture instruction (QIAGEN; Cat No.: 79306). The RNA samples were treated with DNase I for 1 h at 37°C and then their concentrations were measured using a spectrophotometer and integrity was ensured through analysis of RNA on a 1% (w/v) agarose gel. The quality of the RNA samples was examined by using Agilent RNA 6000 pico kit with Agilent 2100 bioanalyzer (Agilent Technologies Inc.). After checking the RNA quality, total RNA samples were submitted to the Novogene Genomics Singapore Pte Ltd for RNA deep sequencing (RNA-seq).  The NEBNext Ultra II RNA library kit (New England Biolabs) was used for RNA-Seq library construction. Fragmented poly A+ RNA samples were converted to cDNA by random primed synthesis using ProtoScript II reverse transcriptase (New England Biolabs). After second strand synthesis, the double-stranded DNAs were treated with T4 DNA polymerase, 5' phosphorylated and then an adenine residue was added to the 3' ends of the DNA. Adapters were then ligated to the ends of these target template DNAs.  After ligation, the template DNAs were amplified (5-9 cycles) using primers specific to each of the non-complimentary sequences in the adapters. This created a library of DNA templates that have non-homologous 5' and 3' ends.  A qPCR analysis was performed to determine the template concentration of each library. The PCR-based cDNA libraries were sequenced using Illumina NovaSeq 6000 (PE150) technology with the insert size between 250-300 bp.
attributes.tag: GEO Accession
attributes.value: GSM4661641