Computational protocol: PRC2 is dispensable for HOTAIR‐mediated transcriptional repression

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Protocol publication

[…] Total RNA from MDA‐MB‐231 cell lines was isolated by TRIzol extraction and quality‐verified by Bionalyzer. Isolated RNA was used to prepare cDNA libraries and amplified with primers containing sequences required for the Illumina platform. PCR products were cleaned and subjected to 100‐bp paired‐end sequencing on an Illumina Hi‐seq 2500. Sequenced reads from duplicate samples were assembled on the human genome hg19, using tophat_2.0.6 (Kim et al, ).The Htseq software (v0.6.0.) was used to define the number of reads associated with each gene. TMM normalization from the edgeR package v3.6.2 (Robinson & Oshlack, ) was first applied. As described in the guideline of limma R package v3.20.4, normalized counts were processed by the voom method (Law et al, ) to convert them into log2 counts per million with associated precision weights. The differential expression was estimated with the limma package. The P‐values were adjusted for multiple testing using the Benjamini–Hochberg procedure. Finally, differentially expressed genes with a log fold change > 1, FPKM > 1, and adjusted P‐value < 0.05 were used for downstream analysis. Genes' FPKM was estimated using the Cuffquant and Cuffnorm tools of the Cufflinks suite (v2.2.1).The hierarchical clustering was performed using a Pearson correlation distance and a Ward linkage (R v3.2.0, hclust function). [...] SHAPE‐MaP structure probing was performed as described by Smola et al (). Refolded RNA was incubated with 10 mM 1M7(+) (1‐methyl‐7‐nitroisatoic anhydride), 10 mM NMIA (+) (N‐methylisatoic anhydride) or an equal amount of pure DMSO as a control (−) for 3 min or 22 min at 37°C, respectively, due to the different half‐lives of the SHAPE reagents. The samples were then purified by G50 columns and subsequently fragmented to obtain 300‐bp RNA fragments. Reverse transcription was then performed in the presence of Mn2+ using SuperScript III reverse transcriptase kit (Invitrogen); finally, samples (+) and (−) were purified using G50 columns. In parallel, an RNA‐denatured sample was treated following the same steps as the (+) samples as a second negative control (−). SHAPE reactions (+) and (−) were then sequenced using an Ion Torrent sequencing platform, and sequencing data were taken into a bioinformatics pipeline to obtain SHAPE reactivities for 1M7 or NMIA for each RNA nucleotide and normalized for DMSO negative control and RNA‐denatured negative control. The bioinformatics script provided by the Weeks laboratory was adapted for Ion Torrent output files by A. Saadi and Y. Ponty (manuscript in preparation).To generate the HOTAIR secondary structure maps using the software RNAstructure, SHAPE 1M7 reactivity was used to provide pseudo‐energy constraints, while VARNA software was used to visualize the predicted structure (Darty et al, ). Resulting structures were manually evaluated for match with NMIA probing data. SHAPE reactivities are listed in the source data for Fig C. […]

Pipeline specifications

Software tools RNAstructure, VARNA
Application RNA structure analysis