Computational protocol: Bromodomain Protein BRD4 Is a Transcriptional Repressor of Autophagy and Lysosomal Function

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Protocol publication

[…] KP-4 cells were transfected with Control #1, BRD4 #1, or BRD4 #2 siRNA for 72 hr. At 72 hr after transfection, total RNA was isolated and purified using RNeasy mini kit (QIAGEN, Cat#: 74104). Quality of the purified RNA was assessed using an Agilent RNA 6000 Nano kit and 2100 Bioanalyzer. Libraries for cluster generation and DNA sequencing were prepared following an adapted method from () using TruSeq RNA Sample Prep Kit v2 (Illumina, Cat#: RS-122-2001). Quality and quantity of the DNA libraries were assessed on an Agilent 2100 Bioanalyser and Qubit (Thermo Fisher Scientific), respectively. The libraries were run on the Illumina Next Seq 500 using the High Output 75 cycles kit (2x36 cycles, paired end reads, single index). The results were then analyzed as follows. Quality checks on the raw RNA-Seq data files were conducted using fastqc (https://www.bioinformatics.babraham.ac.uk/projects/fastqc/) and fastq_screen (http://www.bioinformatics.babraham.ac.uk/projects/fastq_screen/). RNA-Seq reads were aligned to the GRCh37 () version of the human genome using tophat2 version 2.0.10 () with Bowtie version 2.1.0 (). Expression levels were determined and statistically analyzed by a combination of HTSeq version 0.5.4p3 (http://www-huber.embl.de/users/anders/HTSeq/doc/overview.html), the R 3.1.1 environment, utilizing packages from the Bioconductor data analysis suite and differential gene expression analysis based on a generalized linear model using the DESeq2 (). Enrichment analysis for Gene Ontology terms within this gene set was performed using g:Profiler (). [...] Quantification of western blotting was performed using ImageJ64 software (https://imagej.nih.gov/ij/) using Gel analyzer script. Signal intensity of the protein of interest was normalized to that of loading control (GAPDH, Hsp90, or β-actin). [...] The number of LC3 and WIPI2 puncta were counted using CellProfiler software (http://cellprofiler.org) and normalized to the number of nuclei. The area of LAMP-1-, LysoTracker Red-, and Magic Red CathepsinB-positive compartments was measured using ImageJ64 software (https://imagej.nih.gov/ij/) and normalized to the number of nuclei. […]

Pipeline specifications

Software tools FastQC, FastQ Screen, TopHat, Bowtie, HTSeq, DESeq2, g:Profiler
Application RNA-seq analysis
Diseases Carcinoma