Nebnext mrna second strand synthesis module

For RNA-seq, RNA was extracted from cells used for dCLIP experiments. Starting amount of Total RNA was 4µg. RNA was depleted of ribosomal RNA using Ribominus kit (Life technologies). Strand-specific cDNA libraries were constructed using Superscript III reverse-transcriptase for first-strand synthesis, NEBNext mRNA Second Strand Synthesis Module supplemented with dUTP (NEB) for second-strand synthesis, and NEBNext ChIP-Seq Library Prep Master Mix Set for library preparation. Libraries were subjected to high-throughput sequencing using Illumina HiSeq 2000 apparatus according to manufacturer instructions. Approximately 40 million single-end 50nt reads were generated for every RNA-seq sample (Table S3). Data processing was performed essentially as described previously (Kung et al., 2015 (link)). Adaptor sequences were trimmed from libraries with Trim Galore! v0.3.3 (for dCLIP-seq and RNA-seq; stringency 15). PCR duplicates were removed by custom programs prior to alignment. To account for the M. mus (mus) / M. castaneus (cas) hybrid character of the ES cell lines, reads were first aligned to custom mus/129 and cas genomes, and then mapped back to the reference mm9 genome. RNA was aligned with Tophat (v2.0.8 or greater). Post-processing of mm9 alignments was performed with custom C and Perl programs and bash shell scripts, SAMtools v0.1.18, and BEDtools v2.17.0.

Total RNA extracted from 40μm thick sections of FFPE tumor was reverse-transcribed with random hexamer primers using the SuperScript® III First-Strand Synthesis System (Invitrogen). Double stranded cDNA was synthesized with the NEBNext® mRNA Second Strand Synthesis Module (New England Biolabs).^{35 (link)} Hybrid selection of indexed, adaptor-ligated libraries was performed using the cDNA Kinome hybridization kit with 612 transcripts of kinases and kinase-related genes (Agilent SureSelect Human Kinome Kit).^{36 (link)} Selected libraries were sequenced on the HiSeq-2000 instrument (Illumina) with 49 x 49 paired reads. For RNA sequencing, we used a sequencing approach targeting 612 transcripts of kinases and kinase-related genes. We aimed for a high number of unique read pairs (≥ 50,000,000) per sample (Supplementary Table S6).

RNA-seq experiment was performed as previously reported (11 (link)). In vivo bone lesions were collected 4 weeks after inoculation. BICA samples were harvested and pooled 3 weeks after tumor seeding. All specimens were grinded and homogenized by Precellys 24 Homogenizer (Bertin instrument) with 2.8mm stainless steel beads in 2mL reinforced tubes (Bertin instruments, MK28R). Total RNA of cancer/bone mixture was extracted using the Direct-zol RNA miniprep kit (Zymo Research, R2051). The first cDNA strands were prepared by RevertAid First Strand cDNA Synthesis Kit (Thermo Scientific, K1622) with > 200 ng total RNA input. The second cDNA strand synthesis was performed with NEBNext mRNA Second Strand Synthesis Module (NEB, E6111L). Libraries were generated with the Illumina Nextera XT DNA Library Prep Kit (Illumina). Cluster generation was conducted with the Illumina Nextseq 500/550 high output v2 kit and sequenced on the Illumina Nextseq 500 equipment with the assistance of BCM Genomic and RNA Profiling Core. The RNA-seq data were analyzed with the assistance of Roswell Park Bioinformatics Resource.