CpBV-H4 ChIP-Seq
CpBV-H4 ChIP-Seq
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. 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. 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 The precipitated DNA was dissolved in Tris-EDTA buffer and read by an Illumina using paired end sequencing.
Lab of Insect Molec Physio College of Natural Sciences,Andong National Univ
E-MTAB-1835:T-CHIP-DNA