Nextseq 500 sequencing device

RNA-seq library preparation was performed with Illumina’s Stranded mRNA Prep Ligation Kit following the Stranded mRNA Prep Ligation Reference Guide (June 2020) (document no. 1000000124518 v00). Libraries were profiled on a 2100 Bioanalyzer (Agilent Technologies) and quantified using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Q32851) in a Qubit 2.0 Fluorometer (Life Technologies), following the manufacturer’s recommended protocols. Samples were pooled in equimolar ratios and sequenced on an Illumina NextSeq 500 sequencing device with one or two dark cycles upfront as 79-, 80- or 155-nt single-end reads.

Whole-exome sequencing (WES) using the TruSeq DNA Exome Kit, the NextSeq 500/550 Mid Output Kit v2.5, and a NextSeq 500 sequencing device (all Illumina, CA, USA) was done in all three cases. Input material was 400 ng of DNA obtained from the peripheral blood (for germline exome) and formalin-fixed, paraffin-embedded (FFPE) tumor sample with ≥20% cancer cell count measured in the surface area of tissue slides for somatic exome. WES was done with high coverage where at least 90% of targeted regions were covered 20 times.

Constructs were snap frozen in liquid nitrogen and stored at -80°C. RNA was extracted using Trizol in a Cryo-Cup grinder (BioSpec products, Bartlesville, UK) on dry ice. Total RNA was prepared with the Direct-Zol RNA miniprep kit and RNA clean and concentrator (Zymo Research, Cambridge, UK) according to the manufacturer’s instructions. Libraries were prepared using the NEBNext Poly(A) mRNA magnetic isolation module (New England Biosystems, Ipswich, UK) and then the NEBNext ultra-directional RNA library prep kit was used to create the final library. The sample was sequenced using 41bp paired end configuration with an Illumina NextSeq 500 sequencing device. Reads were trimmed using trimmomatic (20 (link)), pseaudoaligned using kallisto (21 (link)) with an index built from the hg38 cDNA fasta reference sequence, and then quality of the pseudoalignment was assessed using FastQC. Differential gene expression was performed using the sleuth package. Genes were considered differentially regulated based on an adjusted p value < 0.01. Differentially expressed genes between two groups (constructs at 0 weeks and after 8 weeks of treatment with osteogenic medium) were analyzed for enriched terms using GSEA in R. Heatmaps were generated in R using ggplot2. GO and KEGG (GSEA) analysis was performed on WebGestalt.