title: GSM5466218: ACP4_n; Diaphorina citri; RNA-Seq
library_construction_protocol: Freeze-dried HLB-infected midribs (from 15–25 leaves) were ground to a fine powder in liquid nitrogen. 1ml of TLE extraction buffer was added to 100 mg citrus powder or the pellet obtained after the above bacterial isolation. Then, 60 µL of 500 mM CaCl2 was added, mixed thoroughly, and placed on ice for 10 min. Centrifugation at 12,000 x g for 10 min at 4 °C was performed, followed by two rounds of separation with 500 µl of phenol:Chloroform:isoamyl alcohol (25:25:1) and centrifugation at 15,000 x g for 10 min at 4 °C. The supernatant was mixed with 2 M LiCl and incubated overnight at -20 °C. RNA was precipitated by centrifugation at 15,000 x g for 30 min, washed twice with ice-cold 75% ethanol, centrifuged at 15,000 x g for 15 min and re-suspended in 20 µl of RNAse-free water. 10–15 adult psyllids were ground in liquid nitrogen, immediately suspended in 50 μl TRIzol® (Invitrogen) and ground until complete homogenization. An additional 950 μl of TRIzol® was added to the homogenate, followed by separation with 200 µl of chloroform, precipitation with 500 µl of isopropanol, and two washes with 75% ethanol as indicated by the manufacturer. The pellet was resuspended in 20-30 µl of RNAse-free water. RNA was converted into sequencing-ready libraries for Illumina sequencing using TruSeq Stranded Total RNA reagents. Poly(A) Polymerase (NEB) treatment (polyA) and Poly(dT)-depleted library construction was tested in parallel. cDNA libraries were subjected to RNASeq, using an Illumina HiSeq-4000 system with 150-bp strand-specific single-end reads with technical duplicates.