title: GSM2044844: Biorep_2_48_hours paired-end; Mamestra configurata nucleopolyhedrovirus A; RNA-Seq
library_construction_protocol: Following removal of the peritrophic matrix and food bolus, the midgut tissue was directly immersed in 0.5 ml of TRIzol (Life Technologies). The midgut samples in TRIzol samples were stored at -70oC until tissue collection was complete. Total RNA was extracted from each pool of 5-6 midguts by homogenizing in 1.0 ml of TRIzol reagent according to the manufacturer's protocol (Invitrogen). Total RNA was resuspended in 100 µl of sterile DEPC-treated H2O and quantified by measuring absorbance at 260 and 280 nm using a Nanodrop spectrophotometer (Fisher-Thermo). The quality of the RNA was assessed using an RNA 6000 Nano Kit and a Bioanalyzer 2100 instrument (Agilent). Libraries were synthesized and sequencing performed at the National Research Council, Saskatoon, Canada. Briefly, libraries were prepared from 1 ug total RNA, representing 6 MacoNPV-A 90/2 infected BAW midguts as pooled samples. For each pooled sample treatment, there were two  biological replicates representing separate infection events collected at 6 time points post-infection plus two controls, water and 0.5% fluorescent brightener 28 (Sigma Chemicals). The TruSeq RNA Sample Prep Kit v2 (Illumina) was used to generate cDNA libraries for polyA-selected RNA from each time point.  The quality of each library was checked on a DNA 1000 chip on the 2100 Bioanalyzer (Agilent Technologies Inc.) and the concentration was determined by qPCR using the KAPA SYBR®FAST qPCR Kit (Kapa Biosystems) on the StepOnePlus Real-Time PCR System (Applied Biosystems). The cDNA library insert sizes ranged from 200-600 bp with an average size of 275 bp, including the adapter sequences (126 bp), therefore the insert size range was ~75-475 bp. The samples were multiplexed at 14 libraries/lane.  Equimolar concentrations of the libraries were pooled and a final concentration of 10 pM was used for clustering in one lane of a flowcell on a cBOT (Illumina).   The samples were sequenced (2 x 101 cycles, paired-end reads) on the HiSeq2500 (Illumina) using the TruSeq SBS Kit v3-HS 200 cycles Kit (Illumina). Each cDNA library was sequenced in two separate sequencing runs to increase the number of reads.
attributes.tag: GEO Accession
attributes.value: GSM2044844