Illumina HiSeq 2000 sequencing; RNAseq of ZC3H5 interacting mRNA
RNAseq of ZC3H5 interacting mRNA
RNA IP (Flow_Through or Elute) To identify the transcripts bound to ZC3H5, 2x109 cells  expressing TAP-ZC3H5 were washed in ice-cold Voorheis's-modified phosphate-buffered saline (vPBS; PBS supplemented with 10 mM glucose and 46 mM sucrose) and the cell pellet snap frozen in liquid nitrogen. The RNA immunoprecipitation was done essentially as described in Mugo et al. (PMID: 32221926). Briefly, the cell extracts were incubated with IgG-coupled magnetic beads (NEB) for 2 h at 4°C, and the unbound fraction was collected. After extensive washing, bound ZC3H5 was eluted using TEV protease for 2 h at 16°C.  Trifast then rRNA depletion by RNAse-H treatment. RNA was isolated using Trifast reagent (Peqlab). Total RNA from the unbound fraction was depleted of ribosomal RNA (rRNA) using RNAse H and a mixture of 50-base DNA oligos complementary to trypanosome rRNAs. The recovered RNA from both bound and unbound samples were then analyzed by RNA-Seq (David Ibberson, Bioquant). Illumina HiSeq
Experimental Factor: fraction
eluate