title: A. argentata Tubuliform silk gland 2 RNAseq
design: Adult female Argiope argentata were collected in San Diego County, CA, USA. Tubuliform silk gland tissue was dissected and flash frozen in liquid nitrogen. Four individuals were dissected. Tubuliform silks glands from two individuals were combined to make tubuliform silk gland biological replicate 1 (Tub_R1). The corresponding tissues from two other individuals were used for the second biological replicate (Tub_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 from the tubuliform silk glands was processed into cDNA using the Ovation 3’ DGE System (NuGen, San Carlos, CA, USA). The Ovation 3’ DGE System generates double-stranded cDNA from the 150-500 bases at the 3’end of each transcript. 3’ targeting occurs during first strand synthesis with anchored oligo(dT) primers. After second strand synthesis, 3’ biased cDNA is amplified with the NuGen Ribo-SPIA (Single Primer Isothermal Amplification) technique (Tariq et al. 2011). 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.    The total RNA extracted was also made into an RNAseq libraries 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 libraries were paired-end sequenced for 100 cycles. The 3’ DGE libraries were 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.