GSM6217232: B_mBB_2; Solenopsis invicta; RNA-Seq
Dissected samples were stored in liquid nitrogen. Total nucleic acids from brains were extracted using an Agencourt FormaPure kit (Beckman Coulter Life Sciences, Indianapolis, Indiana) with volumes scaled down by a factor 4. The first two washing steps (wash buffer and 70% ethanol) were carried out twice. Half of the extracted nucleic acids were eluted with 20 µl of RNase free water and concentrated to 5 µl. The sample was digested by adding 20 µl of DNase I mix (60 µl contains 1.5 µl 1M Tris-HCl [pH 7.5], 3.75 µl 0.1M MgCl2, 1.5 µl 2.5 M KCl, 1.5 µl 0.1M DTT, 3.75 µl RNasin Plus [40u/µl; Promega, Madison Wisconsin], 5 units of DNase I [RNase-free; cat no. 04 716 728 001; Roche, Basel, Switzerland] and 47.5 µl RNase-free water) and incubating at 37°C for 15 min. Then 25 µl of T.E. buffer (pH 7.5) and SDS (0.2% final concentration) were added and RNA was extracted with chloropane. 8 µl of linear acrylamide (5 mg/ml; Ambion; Austin, Texas) was added as carrier to the aqueous phase and nucleic acids were precipitated by adding sodium acetate to 0.3 M and 2.5 volumes of ethanol followed by a wash in 70% ethanol. The pellet was air-dried for 5 min. and re-suspended in 9 µl of RNase-free water. Sequencing libraries for brains were prepared using SMARTer Ultra Low Input RNA for Sequencing-V3 kit (Takara Bio, Kusatsu, Japan) with a modified protocol provided by manufacturer for brains. After the fragmentation step (volume 75 µl), 8 µl of acrylamide carrier was added and double-strand cDNA was precipitated with ethanol followed by a wash in 70% ethanol. The pellet was air-dried for 5 min., re-suspended in 10 µl 10 mM Tris-HCL (pH 8.5) and processed further with the sequencing kit. Sequencing libraries for gasters were prepared using the KAPA stranded RNA-seq kit (Kapa Biosystems, Wilmington, Massachusetts) following manufacturer's instructions.