title: Argiope argentata cephalothorax 3' DGE 2
design: Adult female Argiope argentata were collected in San Diego County, CA, USA. Cephalothorax (head-body, non-silk gland control) was dissected and flash frozen in liquid nitrogen. Four individuals were dissected. Cephalothorax tissues from two individuals were combined to make cephalothorax biological replicate 1 (Cep_R1). The corresponding tissues from two other individuals were used for the second biological replicate (Cep_R2).  Total RNA was extracted from tissue homogenized in TRIzol (Invitrogen, Carlsbad, CA, USA) and purified with an RNeasy Mini kit (Qiagen, Valencia, CA, USA). Carryover genomic DNA was removed with Turbo DNase (Life Technologies, Grand Island, NY, USA). Each RNA extraction was quantified with a Nanodrop ND-1000 (ThermoFisher Scientific, Waltham, MA, USA) and examined for integrity with a Bioanalyzer (Agilent 2100, Santa Clara, CA, USA). Samples were stored at -80°C.   Total RNA extractions were processed into cDNA using the Ovation 3’ DGE System (NuGen, San Carlos, CA, USA). The resulting cDNA libraries were purified with the AccuPrep PCR Purification Kit (Bioneer Inc., Alameda, CA, USA). For quality control and to estimate fragment size and yield, an aliquot of each sample was visualized with an Agilent 2100 Bioanalyzer (Agilent technologies, Santa Clara, CA, USA). Samples were stored at -20 ºC prior to proceeding. Illumina-compatible library adaptors were attached to the 3’ DGE libraries with the Encore NGS Mulitplex System I (NuGen). Libraries were quantified with a Nanodrop ND-1000 and molarities determined with the Bioanalyzer.   Total RNA from the cephalothorax tissue replicate with the higher RNA yield (Cep_R2) was made into an RNAseq library with the TruSeq RNA Sample Prep Kit v2 (Illumina) by the Johns Hopkins University School of Medicine Deep Sequencing and Microarray Core.   Libraries were sequenced on a HiSeq2500 platform (Illumina) at the University of California, Riverside Institute for Integrative Genome Biology. The RNAseq library  was paired-end sequenced for 100 cycles. The 3’ DGE libraries (Cep_R1 and R2) was single-end sequenced for 50 cycles. The biological replicates of the 3’DGE libraries were run in separate lanes, and each 3’DGE library was run twice, generating technical replicates.