Instrument: Illumina HiSeq 2000
Strategy: OTHER
Source: TRANSCRIPTOMIC
Selection: other
Layout: SINGLE
Construction protocol: Cultures were incubated for 5 min in 500 ml 37ºC RPMIc, 100µg/ml cycloheximide (Acros Organics) and harvested by centrifugation for 8 min at 3.65 xg at room temperature. An aliquot was removed and flash frozen in lN2 for total total RNA purification and mRNA-Seq library preparation. RBCs were lysed with ice-cold 0.1% saponin in 1X PBS, 100ug/ml cicloheximide. Parasites were resuspended in ice-cold parasite lysis buffer (15 mM KOAc, 15 mM MgOAc, 10 mM Tris HCl pH 7.4, 0.5 mM DTT, 0.5% Triton X-100, 100 ug/ml cyclohexamide) and dripped into a conical tube filled with, and immersed in, lN2. Frozen cells transferred placed in lN2 pre-chilled chambers and pulverized for 3 min at 15 Hz, on a Retsch MM301 mixer mill. Pulverized cells were thawed on ice and cell debris was removed by centrifugation at 4ºC, 16000 xg for 10 min. The polysome-containing supernatant was treated with 2.88 U/ug Micrococcal nuclease for 30 min at room temperature and immediately loaded onto sucrose gradients. Ribosome footprints were extracted from sucrose gradient monosome peak fractions. Libraries were prepared as in Ingolia et al. Science 2009