Computational protocol: The IAP family member BRUCE regulates autophagosome–lysosome fusion

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Protocol publication

[…] To prepare RNA-Seq samples, total RNA was isolated from MEFs expressing shRenilla and shBruce#1, starved for 2 h or grown in regular media using TRIzol (Life Technologies, 15596026). Contaminating DNA was digested by TURBO DNA-free Kit (Ambion, AM1907) and Bioanalyzer 2100 (Agilent Technologies) was used to determine the quality and quantity of RNA according to the manufacturers’ instructions. The library was prepared from these samples by poly(A) enrichment (New England Biolabs, Ipswich, MA). The resulting fragmented samples were sequenced on a HiSeqV4 SR50 with a read length of 50 (by VBCF-NGS). The reads were mapped to the Mus musculus mm10 reference genome with STAR (version 2.4.0d). Reads aligning to rRNA sequences were filtered out prior to mapping. The read counts for each gene were detected using HTSeq (version 0.5.4p3). The counts were normalized using the TMM normalization from edgeR package in R. Before statistical testing, the data was voom transformed and then the differential expression between the sample groups was calculated with limma package in R. The functional analyses were done using the topGO and gage packages in R. For visualization, heat maps were created using R and fragment alignments were processed using the Integrative Genomics Viewer (IGV_2.3.40 software), […]

Pipeline specifications

Software tools STAR, HTSeq, edgeR, limma, TopGO, IGV
Application RNA-seq analysis