Computational protocol: The role of the zinc finger protein ZC3H32 in bloodstream-form Trypanosoma brucei

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Protocol publication

[…] To study interactions of ZC3H32 we used bloodstream-form trypanosomes in which one ZC3H32 allele was knocked out and the other bore a sequence encoding an N-terminal TAP tag. For protein partners, TAP-ZC3H32 was purified on an IgG column, bound protein was eluted with Tobacco Etch Virus (TEV) protease, bound to a calmodulin column and eluted with EGTA [, ]. Proteins were subjected to denaturing SDS-PAGE and analysed by mass spectrometry. For RNA binding, only the first purification step was applied. The bound and unbound RNAs were purified. For RNASeq, RNA preparations were rRNA-depleted using rRNA-complementary oligonucleotides and RNase H [], except with the 24h RNAi experiment which was done using poly(A)-selected RNA. Library-building and sequencing were done using standard Illumina protocols (see E-MTAB-5612 and E-MTAB-4558). The reads were aligned using Bowtie2, allowing for up to 20 alignments and one mis-match, then all reads aligning to open reading frames were counted using custom scripts []. The data were analysed using DeSeq2 [] Automated scripts are available for both the sequence alignments [] and DeSeq2 analysis, including functional class enrichment []. […]

Pipeline specifications

Software tools Bowtie2, DESeq2
Application RNA-seq analysis