Heliothis virescens hemocyte cDNA library
Heliothis virescens eggs were received from the North Carolina State University, Dept. of  Entomology Insectary (Raleigh, NC, USA). Larvae were reared individually on an artificial wheat  germ based diet (Bio-Serv, Frenchtown, NJ, USA) under standard conditions of 14 h:10 h (L:D)  photoperiod, 55% RH, 28 °C. Synchronized early-4th instar larvae (4 hours post molt) were either  mock-infected with a control inoculum absent virus or infected with an LC99 dose of HzSNPV per os  (5 × 108 polyhedra/mL). To activate the antibacterial immune response early 5th instar larvae  were punctured with a tungsten needle dipped into a 1 μg/mL suspension of lipopolysaccharide and  peptidoglycan (Sigma Chem. Co., St. Louis, MO, USA) in PBS. The antifungal response was  activated by puncture with a tungsten needle dipped into a 1 μg/mL suspension of β-glucan, curdlan  and laminarin (Sigma Chem. Co., St. Louis, MO, USA) in PBS. Hemocytes were collected at 12 hpi in  order to capture early host-responses to HzSNPV infection. Hemocytes were collected from 30 insects  from each treatment according to previously published methods. Larvae were bled through a  punctured anterior proleg into ice cold PBS containing a crystal of phenylthiourea to prevent  melanization. Hemocytes were pelleted by centrifugation at 5000 × g for 4 min, plasma supernatant was  removed, and stored at −85 °C for later use. Total RNA was extracted from hemocytes using RNeasy™  kits (Qiagen, Valencia, CA, USA).   Hemocyte RNA pools were submitted to University of Missouri, Bond Life Sciences Center DNA  Core for Illumina GAII sequencing. RNA quality of the pooled time points was examined using the  Experion Automated Electrophoresis system (Bio-Rad, Hercules, CA, USA). Libraries were  constructed according to the standard Illumina RNA-seq protocol (Part# 1004898 Rev. A, rev Sept 08)  for the pooled PCR products except for the fragmentation step. First strand and second strand  cDNA synthesis was carried out with Invitrogen superscript II reverse transcriptase and random hexamer primers according to the manufacturers protocol. Illumina paired-end adapters were ligated  and a gel size-selected ~300 bp fragments depleted of adapter dimer and large chimeric fragments  were subjected to 15 rounds of selective PCR enrichment. The libraries were run on an Illumina GAIIx sequencing instrument.