title: GSM4969136: Day 0 Brain replicate 5 [d0_5_br]; Harpegnathos saltator; RNA-Seq
library_construction_protocol: For extracting RNA, a pipette was used to gently aspirate and agitate the tissue. After agitation, fresh Trizol was added to bring the volume to 1 ml. The mixture was placed into a new 1.5 mL microcentrifuge tube and 200 μl chloroform per 1ml Trizol was added. The tube was inverted repeatedly for 30 sec and left at room temperature for 5 min. The Trizol mixture was centrifuged for 15 min at 12,000x g at 4oC. The top clear aqueous phase (~500 μl) was transferred to a new microcentrifuge tube containing 1μl of Glycoblue (ThermoFisher, #AM9515) and 1x volume of isopropanol. The RNA/isopropanol/Glycoblue mix was then centrifuged for 30 min at 20,000x g at 4°C. The supernatant was removed and the pellet was washed twice with 70% and 80% ethanol, respectively and subsequently centrifuged for 10 min at 20,000x g at 4°C. The pellet was air dried for 5 min before dissolved in BTE buffer (10mM Bis-Tris pH 6.8, 1mM EDTA). RNA samples were treated by DNase I (Ambion, #2239) and DNase I was removed by phenol/choloform extraction and isopropanol precipitation. For library preparation, polyA+ RNA was isolated from approximately 500 ng total RNA using NEBNext ® Poly(A) mRNA Magnetic Isolation (E7490). Libraries were prepared using NEBNext® Ultra II RNA library prep kit (E7770, E7335 and E7500), quantified before and after amplification using KAPA SYBR® Fast universal qPCR Kit (KK4854) and sequenced on Illumina Hiseq 2500.