title: GSM6217254: B_pBL_3; Solenopsis invicta; RNA-Seq
library_construction_protocol: 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.