Computational protocol: A heterochromatin-dependent transcription machinery drives piRNA expression

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Protocol publication

[…] Co-immunoprecipitated proteins were subjected to on-bead digestion with LysC and elution with glycine before digestion with Trypsin. The resulting peptides were analyzed using a Dionex UltiMate 3000 HPLC RSLC nano system coupled to a Q Exactive mass spectrometer equipped with a Proxeon nanospray source (Thermo Fisher Scientific). Peptides were eluted using a flow rate of 230 nl min-1, and a binary 3h gradient, respectively 225 min and the data were acquired with the mass spectrometer operated in data-dependent mode with MS/MS scans of the 12 most abundant ions. For peptide identification, the RAW-files were loaded into Proteome Discoverer (version 2.1.0.81, Thermo Scientific) and the created MS/MS spectra were searched using MSAmanda v1.0.0.6186 against Drosophila melanogaster reference translations retrieved from Flybase (dmel_all-translation-r6.06). An in-house-developed tool Peakjuggler was used for the peptide and protein quantification (IMP/IMBA/GMI Protein Chemistry Facility; http://ms.imp.ac.at/?goto=peakjuggler). Using custom R scripts, average enrichment between bait and control IP experiments were calculated. Adjusted p values were calculated using the limma R package. [...] RNA-Seq reads were trimmed to high quality bases 5-45 before mapping to the Drosophila genome (dm6, r6.10) using STAR or to Drosophila melanogaster transposon consensus sequences using SALMON. For genomic mapping by STAR normalization between samples was done based on the number of genome-unique mapping reads for each sample. Subsequent quantification of reads mapping to 1 kb tiles was done using bedtools, while relative quantification and plotting was done in R. Briefly, RNA-seq tile signal was normalized to the estimated mappability scores for each 1 kb window. A pseudo-count of 1 was then added to each tile value before calculation of log2 fold-change values relative to control genotype samples. […]

Pipeline specifications

Software tools STAR, BEDTools
Application RNA-seq analysis
Organisms Drosophila melanogaster