nucleic acid extraction protocol
Briefly, 100 larvae each of Tribolium castaneum injected with recombinant CpBV-H4 and truncated CpBV-H4 were homogenized in PBS on ice, followed by the addition of formaldehyde to a final concentration of 1% and incubation at 37C for 10 min. Chromatin immunoprecipitation assays were performed using a QuikChIP kit (IMGENEX) according to the instruction manual
high throughput sequence alignment protocol
Reads were mapped to the Tribolium castaneum genome (Tcas_3.0) using Bowtie (version 0.12.7). Up to 2 mismatches in the seed sequence of each read were allowed; non-unique reads were disarded.Options for bowtie were : -S –fr –n 2 –m 1
nucleic acid sequencing protocol
The precipitated DNA was analyzed by using Illumina HiSeq 2000. Paired-end sequencing; according to manufacturers protocols Base calls were done by the Illumina software pipeline using RTA 18.104.22.168 and BCL converted using CASAVA 1.8.2 (configureBclToFastq.pl)
nucleic acid library construction protocol
The precipitated DNA was dissolved in Tris-EDTA buffer and read by an Illumina using paired end sequencing.
A full open reading frame of CpBV-H4 and the truncated CpBV-H4 (without N-terminal 38 amino acids) were cloned into pIB eukaryotic expression vector (Invitrogen, Carlsbad, CA, USA) according to previous studies . The recombinant pIB vector was mixed with Metafectene PRO transfection reagent (Biontex, Planegg, Germany) according to manufacturers instruction. Briefly, 0.5 ug of recombinants were mixed with 3 uL of Metafectene reagent and incubated for 20 min at room temperature to allow DNA-lipid complexes to be formed before injection into hemocoel of late instar larvae of T. castaneum. Glass capillary (World Precision Instruments, Sarasota, FL, USA) injection needles were made using a micropipette puller PN30 (Narishige, Tokyo, Japan). A total 60 nL was injected into the larval hemocoel at the rate 10 nL/sec using a micro-syringe pump controller (WPI) under a microscope (Olympus S730, Tokyo, Japan). After 48 h PI, total RNA was extracted using Trizol reagent (Invitrogen) and followed by reverse transcription using RT-Premix (Bioneer, Daejeon, Korea) with an oligo dT and subsequent RNase H (1 unit/uL) treatment. The synthesized cDNA was used as a template for PCR amplification. Control PCR reaction was performed with RNA extract template, which was used as a template to check absence of DNA contamination.
T. castaneum was reared in a dry and dark condition (a relative humidity 60 plus/minus 5%) at room temperature (25 plus/minus 10C) with wheat flour (Pareve, USA). Fully grown late instar larvae (5 mm) were used in this study.