Truseq sequencing adapter

The cDNA library preparation and sequencing for the total RNA-seq analysis were carried out by the Core Unit Systems Medicine (Core Unit SysMed) of the Medical Faculty of the University of Würzburg and the Interdisciplinary Center for Clinical Research of the University Hospital Würzburg. First, ribosomal RNA was removed using RiboCOP META depletion kit followed by ultrasound treatment (one pulse of 30 s at 4°C). An adapter was then ligated to the 3′ end of the fragmented RNA molecules. For the synthesis of first-strand cDNA, the M-MLV reverse transcriptase was used; the introduced 3′-adapter served as a primer. The 5′ Illumina TruSeq sequencing adapters were ligated to the cDNA, and the cDNA was PCR amplified (10–20 ng µL⁻¹). The amplified cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics) and subsequently assessed via capillary electrophoresis. The cDNA was pooled and further purified through preparative agarose gel electrophoresis leading to fragments of cDNA ranging from 200 to 600 bp. The resulting pooled libraries were sequenced using an Illumina NextSeq 2000 system with 100 bp read length.

Total RNAs were isolated from the venom gland tissues by using RNeasy Micro Kit (Qiagen, France) according to the manufacturer’s instructions. Sequencing and cDNA library preparation were performed using Beckman Coulter Genomics services (http://www.beckmangenomics.com/). The mRNA extracted from the venom gland tissues was reverse transcribed into cDNA and amplified using the Ovation RNA-Seq System V2 kit (NuGEN Technologies Inc., USA). After cDNA fragmentation, end-repair and purification were performed using the Agencourt AMPure XP kit (Agencourt Bioscience, Beckman Coulter, San Carlos, CA, USA), and TruSeq sequencing adapters (Illumina, USA) were ligated to cDNA fragments. Finally, the library was PCR-amplified (14 cycles) to about 30 ng/μl by using a high-fidelity DNA polymerase (Beckman Genomics, USA). Illumina TruSeq adapters were ligated to the 5′ and 3′ ends of the cDNA of both samples. The cDNA was finally amplified by PCR using a proofreading enzyme (Beckman Genomics, USA). For Illumina sequencing, the cDNA samples were fractionated on agarose gels ranging in the size from 300 to 500 bp. PCR amplification was designed for TruSeq sequencing using the HiSeq4000 technology according to the instructions of Illumina.

RNA deep sequencing of whole cells was as described (21 (link)). For RNA deep sequencing of ribosome fractions first-strand cDNA synthesis was primed with a N6 randomized primer. After fragmentation, the Illumina TruSeq sequencing adapters were ligated in a strand specific manner to the 5′ and 3′ ends of the cDNA fragments. This way, a strand specific PCR amplification of the cDNA was achieved using a proof reading enzyme. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter Genomics). The cDNA samples were pooled for near equimolar amounts and single-end sequenced (75 bp) on an Illumina NextSeq 500 system. The cDNA reads were analysed via the RNA-seq workflow within Partek^® Genomics suite 6.6, including a QA/QC step to gauge the sequencing quality. Each sample yielded close to equivalent total reads aligned to the E. coli K-12 reference genome CP009273. The experiments were performed in duplicate. Gene ontology (GO) enrichment analysis was performed using the PANTHER Classification System (22 (link)).

Whole cell total RNA was processed by Beckman Coulter Genomics according to the following steps. Poly(A)+ RNA was isolated from the total RNA samples and fragmented with ultrasound (1 pulse of 15 s at 4°C). First-strand cDNA synthesis was primed with random hexamers. Then, the Illumina TruSeq sequencing adapters were ligated to the 5' and 3' ends of the cDNA. The cDNA was finally amplified with PCR using a proof reading enzyme and between 12 and 14 cycles. The TruSeq barcode sequences which are part of the 3' TruSeq sequencing adapters are included in Supplementary file 2. The cDNA was purified using the Agencourt AMPure XP kit (Beckman Coulter ). For Illumina sequencing, the cDNA samples were pooled in approximately equimolar amounts. The cDNA pool was size fractionated in the size range of 350–550 bp on a preparative agarose gel and size range was verified by capillary electrophoresis (Shimadzu MultiNA microchip electrophoresis system). Finally, 2x150 bp paired-end and strand-specific sequencing was performed with HiSeq SBS kit v4 on a HiSeq 2500 system (Illumina, San Diego, CA). RNA-seq sequencing statistics are summarized in Supplementary file 2.

Total RNAs (tRNAs) from venom glands sample were isolated with RNeasy Micro Kit (Qiagen, France) including an on column DNase digestion whereas total RNAs from ant body carcasses were extracted using 400 μl of TRI reagent (Sigma) according to the manufacturer’s protocol. Sequencing and cDNA library preparation were performed by Beckman Coulter Genomics services (http://www.beckmangenomics.com/). Given the very limited amount of the total RNA extracted from the venom glands (7 ng/μl), mRNA from this sample (sample G) was transcribed into cDNA and amplified using the Ovation RNA-Seq System V2 kit, especially applied to limited biological material (NuGEN Technologies Inc.). After cDNA fragmentation, end-repair and purification with the Agencourt® AMPure® XP kit (Agencourt Bioscience, Beckman Coulter, San Carlos, CA, USA), TruSeq sequencing adapters (Illumina) were ligated to the cDNA fragments. Finally, the library was PCR-amplified (14 cycles) to about 20–30 ng/μl using a high fidelity DNA polymerase. For the total RNA sample from ant carcasses (Sample F), poly (A) RNA was isolated and fragmented. First-strand cDNA synthesis was primed with an N6 randomized primer.