Computational protocol: Cathepsin-L Can Resist Lysis by Human Serum in Trypanosoma brucei brucei

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Protocol publication

[…] The RNAi cassettes remaining in the NHS-selected library were specifically amplified from genomic DNA using the LIB2f/LIB2r primers producing a ladder of bands ranging in size from 0.25–1.5-kbp following agarose gel-electrophoresis (data not shown). High-throughput sequencing of the amplified DNA was carried out on an Illumina platform (Beijing Genome Institute). Using paired 150-bp sequencing reads; presence or absence of a 14-bp RNAi-construct signature was recorded in the FASTQ header line. Sequence reads were then trimmed to remove lower-quality sequences and mapped to the T. b. brucei reference genome (release 4.2) using bowtie . BAM files were processed using the SAMtools bioinformatics suite . The maps were explored visually in Artemis, and plots were derived using the Artemis graph tool and processed in Adobe Photoshop Elements 8.0. Stacks of reads that included the 14-bp signature on the positive strand were used to define RNAi target fragment junctions and to assign high-confidence hits as those identified by >1 RNAi target fragment. […]

Pipeline specifications

Software tools Bowtie, SAMtools
Application Non-coding RNA analysis
Organisms Trypanosoma brucei, Homo sapiens
Diseases Trypanosomiasis, African
Chemicals Cysteine