GSM8197054: embryos_dsGFP_24hr_R2; Anopheles gambiae; RNA-Seq
Embryos were collected at different time points after oviposition from control and TL2-depleted blood fed An. gambiae females in four biological replicates and stored in -80C until the day of RNA extraction. After homogenization in a bead-beater, RNA was extracted according to manufacturer's instruction, with mnor modifications. RNA pellets were washed three times with 75% ethanol in RNase-Free water and air-dried for 7-8 minutes. Pellets were resuspended in heated water for 10 minutes, DNase-treated using Turbo DNase according to manufacturer's instructions (Thermo Fisher Scientific), and then purified using PureLink RNA purification mini kit according to manufacturer's instructions (Invitrogen). Libraries were prepared using a SciClone G3 NGSx workstation (Perkin Elmer) using the Kapa mRNA HyperPrep kit (Roche Applied Science). Polyadenylated mRNAs were captured using oligo-dT-conjugated magnetic beads (Kapa mRNA HyperPrep kit, Roche Sequencing) from 300 ng of total RNA on a Perkin Elmer SciClone G3 NGSx automated workstation. Poly-adenylated mRNA samples were immediately fragmented to 300-400bp using heat and magnesium. First-strand synthesis was completed using random priming followed by second-strand synthesis and A-tailing. A dUTP was incorporated into the second strand to allow strand-specific sequencing of the library. Libraries were enriched and indexed using 11 cycles of amplification (Kapa mRNA HyperPrep kit, Roche Sequencing) with PCR primers, which included dual 8bp index sequences to allow for multiplexing (IDT for Illumina unique dual 8bp indexes). Excess PCR reagents were removed through magnetic bead-based cleanup using Kapa Pure magnetic beads on a SciClone G3 NGSx workstation (Perkin Elmer). The resulting libraries were assessed using a 4200 TapeStation (Agilent Technologies) and quantified by QPCR (Roche Sequencing). Libraries were pooled and sequenced on one Illumina NovaSeq S4 lane l using paired-end, 100bp reads.