Computational protocol: RNA immunoprecipitation identifies novel targets of DAZL in human foetal ovary

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Protocol publication

[…] RNA immunoprecipitation was carried out using the Magna RIP™ Kit (Millipore, Livingstone, UK) according to manufacturer's instructions. Immunoprecipitations were performed in lysates made from 2 homogenised 17-week human foetal ovaries, from different fetuses. Separate lysates were made for 3 biological replicates, thus 12 ovaries from a total of 6 fetuses were used for these experiments. RNA–protein complexes were immunoprecipitated using an antibody against human DAZL (dilution 1:100) (#8042, Cell Signalling Technology, Leiden, The Netherlands) (see for antibody validation) or a control matched-IgG. Following RNA purification, the Ovation® RNA-Seq system V2 (NuGEN, The Netherlands) was utilised to generate sufficient quantities of oligo-T primed cDNA (average size 350 bp), which was then subjected to paired-end RNA sequencing using the Illumina HiSeq platform, performed by Edinburgh Genomics, University of Edinburgh, UK. Paired-end sequencing data were processed using TrimGalore v0.4.1 (http://www.bioinformatics.babraham.ac.uk/projects/trim_galore/) to remove the CTTTGTGTTTGA Ovation RNA-Seq adapter using—stringency 5 and—length 30 command line parameters, then mapped to the hg19 assembly of the human genome and Ensembl transcriptome using Tophat v2.1.0 (http://ccb.jhu.edu/software/tophat) with command line parameters-g 1—mate-inner-dist 260—mate-std-dev 110—no-coverage-search—b2-sensitive. Mapped reads that were primary alignments and overlapped Ensembl transcript co-ordinates were counted using Bedtools intersect v2.25.0 (https://github.com/arq5x/bedtools) in-split mode. Paired reads that mapped within the same transcript were counted as a single read. Read counts were imported into R v3.3.2 (https://www.r-project.org/) and biomaRt v2.30.0 (https://bioconductor.org/packages/release/bioc/html/biomaRt.html) used to map Ensembl transcripts to Ensembl genes using the Homo sapiens GRCh38.p7 data set. Genes with multiple transcripts were collapsed into the transcript containing the most read counts, the total number of reads mapped to Ensembl genes counted. Low abundance genes containing fewer than one count per million mapped reads in at least three samples were discarded. Differential analysis was performed using DESeq2 v1.14.0 (https://bioconductor.org/packages/release/bioc/html/DESeq2.html) using the biological replicate as a blocking factor in the design model to identify differences between control and DAZL immunoprecipitations. False discovery rate (FDR)-adjusted P-values of ≤0.05 were considered significant, and genes significantly enriched more than 4-fold in DAZL relative to control immunoprecipitations were considered DAZL targets. This was filtered further to include only Ensembl genes with an HGNC gene symbol in the bioMart database. Relationships between significantly differentially expressed RNAs were further explored using GeneSetDB () using all genes represented in the control and DAZL immunoprecipitations as the universe. […]

Pipeline specifications

Software tools Trim Galore!, TopHat, BEDTools, DESeq2, GeneSetDB
Application RNA-seq analysis
Organisms Homo sapiens, Mus musculus