RNA sequencing of the amygdala from pigs stunned with different gases

A total of 27 female and castrated male crossbreed pigs ([Danish Landrace x Danish Yorkshire]x Pietrain) were examined in this study. These 27 pigs were a subset of 54 pigs which were part of the project for 'testing inert gases in order to establish replacements for high concentration CO2 stunning for pigs at the time of slaughter (TIGER)'. The animals originated from the same farm and had an average slaughter weight of 96.7 +/- 7.2 kg. The animals were equally distributed to one of the used gas mixtures, which was either Argon (Ar), a Nitrogen (N2) - Ar mixture with 40% Ar in the mixture or 92% CO2. Stunning was carried out in a Dip-Lift-System in a commercial slaughterhouse, which was equipped with a new and patented gassing technology by Air Liquide Deutschland GmbH (Krefeld, Germany) to achieve the necessary inert gas concentrations. With this technology a residual Oxygen concentration lower than 1% was achieved for the inert gas mixtures. Each gondola was loaded with 2 pigs as gently as possible. Exposition time was 180 sec for CO2 and 240 sec for Ar to 250 sec for N2 - Ar. After stunning the carcasses entered the normal slaughter routine of the abattoir. After evisceration and splitting the brains of the animals were collected. A tissue block of approximately 25 mm3 was excised from the amygdala 30 min post-mortem and directly stored in RNAlater solution. The locations of amygdalae were identified with the 'Stereotaxic Atlas of the Pig Brain'. Total RNA isolation was performed with the RNeasy Plus Universal Mini Kit (Qiagen N.V., Hilden, Germany). Approximately 20 mg of frozen brain tissue was transferred into a 2 ml tube and mixed with 900 ul QIAzol Lysis Reagent (Qiagen N.V., Hilden Germany) and 5 ul Reagent DX (Qiagen N.V., Hilden, Germany). Additionally, 1.4 mm Ceramic Beads (Biolabproducts GmbH, Bebensee, Deutschland) were added to the mixture and tubes were loaded into a Bead Ruptor Elite (Omni International, Kennesaw GA, USA) and processed in 3 cycles with 4.5 m/sec for 15 sec and a dwelltime for 10sec. Afterwards the samples were processed according to the manufacturer's specifications and eluted in RNAse free water. Samples were diluted to 150 ng/ul for sequencing. RNA sequencing was performed as a service by BGI genomics. Library type was DNBSEQ Eukaryotic Strand-specific mRNA library and sequencing was performed with a DNBseq platform. Paired-end reads with a read length of 100 bp were produced. Raw sequencing reads were filtered and trimmed with SOAPnuke with the following settings: -n 0.001 -l 20 -q 0.4 --adaMR 0.25 --ada_trim --minReadLen 100.