RNA-seq data for virgin adult tissues of CY22B
Samples were flash frozen in liquid nitrogen immediately after dissection, and and stored in 0.2ml Trizol at -80. All samples were homogenized in 0.5ml Trizol Reagent (Invitrogen) with plastic pestle in 1.5ml tube, mixed with 0.1ml chloroform, and centrifuged 12,000g 15min at 4oC, as Trizol RNA extraction protocol. The RNAs in the supernatant about 0.4ml were then collected and purified with Direct-Zol RNA MiniPrep Kit (Zymo), followed the protocol. The total RNAs were eluted in 65μL RNase-Free H2O. About 1μg purified RNAs were treated with 2μL Turbo DNase (Invitrogen) in 65μL reaction, incubated 15min at room temperature with gentle shaking. These RNAs were further purified with RNA Clean and Concentrator-5 (Zymo). One extra wash with fresh 80% ethanol after the final wash step was added into the original protocol. The treated RNAs were eluted with 15μL RNAse-Free H2O, and stored at -80.  The amplified cDNAs were prepared from 100ng DNase treated RNA with Ovation 1  RNA-Seq System V2 (Nugen) and modified protocol. The preparations followed the protocol to the step of SPIA Amplification (Single Primer Isothermal Amplification). The amplified cDNAs were first purified with Purelink PCR Purification Kit (Invitrogen, HC Binding Buffer) and eluted in 100μL EB (Invitrogen). These cDNAs were purified again to 25μL EB with DNA Clean and Concentrator -5 Kit (Zymo) for Nextera library preparation. About 43ng cDNAs were used to construct libraries with Nextera DNA Sample Preparation Kit (Illumina) and modified protocol. After Tagmentation, Purelink PCR Purification Kit with HC Binding Buffer was used for purification and eluted with 30μL EB or H2O. The products (libraries) of final PCR amplification were purified with DNA Clean and Concentractor-5 and eluted in 20μL EB. The average library lengths about 500bp were estimated from profiles of Bioanalyzer (Agilent) with DNA HS Assay. All libraries were normalized to 2-10nM based on real-time PCR method with Kapa Library Quant Kits (Kapa Biosystems). The qualities and quantities of these RNAs, cDNAs and final libraries were measured from Bioanalyzer with RNA HS or DNA HS Assays and Qubit (Invitrogen) with RNA HS or DNA HS Reagents, respectively. Samples were barcoded and sequenced in 4-plex on an Illumina HiSeq 2500 using standard Illumina barcodes. Samples were barcoded and sequenced in 4-plex on an Illumina HiSeq 2500 using standard Illumina barcodes, resulting in high coverage (Figure S4). We sequenced one replicate per sample strain as well as three biological replicates of each reference strain for all tissues. Female tissues for sample strains and one replicate of the reference genome were sequenced with single end reads, while two replicates of reference genome female tissues and all male tissue samples were sequenced with paired end reads.
UC Irvine
CY22B - RNA seq