Functional dissection of the 'kinome' (annotated protein kinase set) in Trypanosoma brucei by RITseq

Kinome-focused RIT-seq: RNAi cell lines were generated as previously described [Jones NG, Thomas EB, Brown E, Dickens NJ, Hammarton TC, Mottram JC. Regulators of Trypanosoma brucei cell cycle progression and differentiation identified using a kinome-wide RNAi screen. PLoS pathogens. 2014;10(1):e1003886. doi: 10.1371/journal.ppat.1003886. PubMed PMID: 24453978; PubMed Central PMCID: PMC3894213]. RNAi lines were pooled, initially into 9 pools each containing 19-25 cell lines and frozen. These pools were then defrosted and further pooled and frozen. This stabilate was utilised in a variety of experiments to determine the role of protein kinases in a range of cell-cycle and DNA-repair conditions (see approrpriate papers for details) To recover the RNAi target sequences from the populations, a single universal primer (5’- TAATGCCAACTTTGTACAAA-3’) was used. This primer was barcoded with 14 different 6-nucleotide tags that permitted combining equal amounts of PCR products in a single sequencing sample. Reads were assigned to each experimental condition later in silico. 10 ng of genomic DNA obtained per sample was PCR- amplified in a 50 µl reaction using Q5® High-Fidelity DNA polymerase (NEB, Ipswich, USA). Groups of 14 barcoded PCR products were pooled in a single sequencing sample, and 400 ng processed according to Illumina Miseq library protocols.