Instrument: Illumina HiSeq X Ten
Strategy: RIP-Seq
Source: TRANSCRIPTOMIC
Selection: other
Layout: PAIRED
Construction protocol: Infected red blood cells (iRBCs) were collected by centrifugation and resuspended in cold PBS(10 volume of iRBC) . Parasites were extracted with 0.15% saponin and washed with cold PBS until the supernatant was clear. Lysis buffer(50 mM Tris-Cl pH7.4, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X-100/ NP-40)with protease inhibitors and RNase inhibitors was added to the parasite pellet[1.5ml/ (1×109 parasites)] and incubated with rotation for 1h at 4℃. Cell debris were spinned out at 13000rpm for 15min at 4℃ and supernatant was incubated with 10ug of anti-Ty1 antibodies at 4℃ for 3h with rotation. For preparation of equilibrated protein-G magnetic beads, 50 ul protein-G magnetic beads were washed once with Wash Buffer(10mM Tris-Cl pH 7.5, 150mM MgCl2, 150mM KCl, 0.1% Triton X-100), then washed once with Elution Buffer, and washed twice with Wash Buffer. Protein-G magnetic beads were mixed with supernatant with protease inhibitors and RNase inhibitors at 4℃ overnight with rotation. Beads were washed twice with 500 ul Wash Buffer and once with PBS and then resuspended in 700ul TRIZOL at 4℃ for 10 min. After removed the beads, 140ul chloroform was added to trizol at room temperature for 5 min and mixture was centrifuged at 13800rpm for 30 min at 4℃. Supernatant was added to a new 1.5ml centrifuge tube and centrifuged at 13800rpm for 2 min at 4℃. Supernatant was mixed with 1ul glycogene and equal volume of isopropanol on ice for 2h. Tube was centrifuged at 13800rpm for 30 min at 4℃. Pellet was washed twice with 1 ml of 75% (vol/vol) ethanol, then air-dried and resuspended in 20µl of RNase-free water[3]. DNase I was used to digest DNA for 15min at room temperature. RNase-free water was added up to 100μl, mixed with equal volume of chloroform/isoamyl alcohol pH<5.0 (24:1), and then mixture was centrifuged at 13500rpm for 5 min. Supernatant was mixed with phenol/chloroform/isoamyl alcohol (24:1), and centrifuged at 13500rpm for 5 min. Supernatant was transferred to a new 1.5ml centrifuge tube with 1ul glycogene, one-tenth volumes of 3 M sodium acetate(pH 5.2), and 2.5 volumes of 100% ethanol and incubated at −80 °C for 20min. Samples were centrifuged at 13500rpm for 15 min and pellet was washed twice with 1 ml of 75% (vol/vol) ethanol, then air-dried and resuspended in 11µl of RNase-free water.