Project description:The first 4 samples belong to the RNA-IP using in situ TAP tagged ZC3H30 in procyclic (insect) form of the parasite T. brucei Lister 427, 2 samples are Elu or eluate, and 2 are FL or flowthrough (unbound) sample. The other 8 samples are also from procyclic cells. 4 samples belong to DKO(ZC3H30 gene double knockout), 2 are non-stressed and 2 are heat shocked samples; the rest 4 samples are DKO-ectopic (ZC3H30 double knockouts, expressing, ectopic copy of ZC3H30) 2 are non-stressed and 2 are heat shocked samples. Heat Shock experiment was done at 39 degree Celsius.
Project description:Exponentially growing cells (OD 0.5-0.8) from the TAP-tagged strain were harvested by ultracentrifugation followed by of washing and flash freezing in liquid nitrogen and breakage in FastPrep24 (ZYmoresearch S6005). The lysate was subjected to IP with Dyna M280 sheep anti rabbit IgG beads for 2h at 4C. An aliquot was taken before the IP as an input control. The IP of the RNA-protein complex was subjected to cleavage by TEV protease to cleave the TAP-tag off the protein. The bound RNA was then precipitated in phenol/chloroform/isoamyl-alcohol and sequenced using 3' T-filling protocol as described by Wilkening et al 2013 to obtain accurate quantification of 3' isoforms. We used the R Bioconductor package DESeq2 (Anders and Huber 2010) to call for differential log change between the Input and the RNA-IP sample and took all those isoforms as differentially regulated which had more than 4-fold change at a FDR of 10% as reported by the package.