title: GSM3903851: PfRrp4_T_rep2_RIP-seq; Plasmodium falciparum; RIP-Seq
library_construction_protocol: An equivalent of 5 x 109 PfRrp6, PfRrp4 and GFP parasites were grown to 15–20 h ring stages in RPMI complete medium. Free parasites were collected by saponin lysis. The resulting parasite pellet was lysed under non-denaturing conditions (50mM Tris-Cl pH7.4, 150mM NaCl, 1mM EDTA, 1mM EGTA, 1% Triton X-100/NP-40, Proteinase inhibitor, RNase inhibitor) for 2h at 4℃ with rotator, then the supernatant was collected by centrifugation at 13,000rpm for 15min at 4℃, and subjected to immunoprecipitation analysis with mouse anti-Ty1 antibodies for 3h at 4 ℃, then incubated with the protein G magnetic beads (Thermo) overnight at 4 ℃. After washing the beads twice with IPP500 (500 mM NaCl, 10 mM Tris-Cl, pH 8.0, 0.05% NP-40, Proteinase inhibitor, RNase inhibitor) and once with 1 x PBS, bounded RNA was eluted by TRIzol reagent for 10min with rotator at 4℃, then RNA was extracted by phenol-chloroform method and treated with DNaseI for 10min at room temperature. IPed RNA was directly used to prepare strand-specific RNA-seq libraries(see below), without poly(A) enrichment. A minimum of two biological replicates was sequenced for each experimental and control immunoprecipitation. The IPed RNA was fragmented to about 300–400 nucleotides (nt) in length, then all subsequent steps were performed in accordance with an KAPA Stranded mRNA-Seq Kit Illumina platform (KAPABIOSYSTEMS KK8421-96 libraries).
attributes.tag: GEO Accession
attributes.value: GSM3903851