GSM2819617: 3D7-IP; Plasmodium falciparum; RIP-Seq
Tandem affinity purification (TAP) of PfPuf3 was performed using the  PfPuf3-PTP parasite line and 3D7 as control.  Parasite pellet was lysed with five-fold volume of pre-cooled PA150 buffer containing a protease inhibitor cocktail. Then the parasites were manually lysed by 60-100 strokes in a Dounce homogenizer with a tight pestle and centrifuged at 16,000 g for 20 min at 4°C. Take 100ul supernatant and labeled as Input, add 1 ml Trizol for future RNA extraction. The rest of supernatant was incubated with 100 ul (settled volume) of IgG agarose beads (GE Healthcare) at 4°C for 2 h. The beads were washed three times with pre-cooled PA150 and equilibrated twice with the TEV buffer. Then the beads were incubated 2 h with 2 ml of TEV buffer containing 100-150 U of TEV protease with gentle rotation at 4°C. The supernatant was obtained by centrifugation at 500 xg for 5 min at 4°C. Add Trizol into the supernatant and then do RNA extraction according to ambion TRIzol Reagent instrction. Libraries were made according to KAPABIOSYSTEMS KAPA Stranded mRNA-seq kit instrction.
GEO Accession
GSM2819617