Ultra directional rna library prep kit

RNA was isolated from FFPE blocks that contained greater than 90% tumor cell content by cutting approximately ten, 8 mm ribbons from each block and isolating the RNA with the RNeasy FFPE Kit (Qiagen). Quantitative real-time RT-PCR amplification was with TaqMan One-Step RT-PCR reagents (ThermoFisher Scientific) and results were normalized to coamplified β-actin. RNA-seq was performed on 2–3 biological replicates. Sequencing libraries were generated using the NEB Ultra directional RNA library prep kit, and we obtained ~25–30 million paired end reads. Additional gene analysis techniques and RNA-seq analysis methods are in Supplementary Materials and Methods. RNA-seq data have been deposited in the National Center for Biotechnology Information (NCBI) Gene Expression Omnibus (GEO) with the accession code GSE109708.

Nuclear RNA was isolated from cell nuclei harvested by hypotonic lysis as described for GRO-seq above. Total RNA was extracted by Trizol™ and libraries generated using the NEBNext® Ultra™ Directional RNA Library Prep Kit. Nuclear RNA did not undergo poly-A selection. Libraries were sequenced for 150 bp of paired-end reads on an Illumina NovaSeq 6000. Analysis of changes in spliced transcripts was made using TopHat v2.1.1 and Cuffdiff v2.2.1 for REFSEQ annotated transcripts from hg38. Because TopHat is optimized to detect splice junctions and small exons, it was inefficient at aligning reads to repetitive elements. To quantify changes to repetitive elements or intronic transcripts, reads were aligned to hg38 using Bowtie2 v2.3.4.1 and compared by FPKM values. Hyperlinks to bam files of SCR or siTDP-treated nuclear RNA-seq data for viewing by genome browser can be copied from here: TopHat alignments (SCR1, SCR2, siTDP1, and siTDP2); Bowtie alignments (SCR1, SCR2, siTDP1, and siTDP2). Fastq files can be downloaded at these hyperlinks: SCR1 forward reads, SCR1 reverse reads, SCR2 forward reads, SCR2 reverse reads, siTDP1 forward reads, siTDP1 reverse reads, siTDP2 forward reads, and siTDP2 reverse reads.

For siKLF5 experiments, BICR31 cells transfected with siNC #1 and siKLF5 #1 (three biological replicates each condition) were maintained in regular media for 2 days before RNA extraction. For KLF5 WT vs. E419Q overexpression experiments, HCC95 cells infected with KLF5 WT and E419Q overexpression constructs (no-tagged, two biological replicates) were maintained in low serum condition (RPMI-1640 media supplemented with 1% FBS), which is consistent with the condition of cell proliferation assays of KLF5 E419Q overexpression, for 2 days before RNA extraction. RNA was extracted using Qiagen RNeasy kit and treated with on-column DNase I. RNA-sequencing (RNA-seq) libraries were prepared using the NEBNext Ultra Directional RNA library prep kit (NEB, E7420S) and sequenced on the Illumina MiSeq instrument (75-bp paired end reads). Sequencing reads were aligned using STAR (119 ) and expression level for each gene was quantified by RSEM (120 (link)). The differential expression analysis was performed using the edgeR and limma pipelines (115 ,121 (link)). The RNA-seq results were uploaded to the Gene Expression Omnibus (GSE88977).