Qiaseq mirna kit

Libraries for whole genome sequencing (WGS) were prepared using the PCR-free KAPA Hyper Prep Kit (Roche). For whole transcriptome sequencing, RNA samples were depleted of the ribosomal RNA using the Ribo Zero Kit (Illumina) and library preparation was performed using the TruSeq Stranded Total RNA Kit (Qiagen). For small RNA sequencing the QIAseq miRNA Kit (Qiagen) was used All library preparation kits were used according to manufacturer’s instructions. Sequencing was performed on a NovaSeq6000 system (Illumina).
For WGS, average coverage for tumor samples was ≥60× and ≥30× for normal samples with a total genomic coverage of ≥95%.
Whole transcriptome sequencing datasets have ≥100 million total reads with less than 20% of ribosomal origin and ≥20 million reads mapping to mRNAs according to Ensembl reference. Ribosomal depletion was performed to remove nuclear rRNA and mt-rRNA.

Libraries for whole genome sequencing (WGS) were prepared using the PCR-free KAPA Hyper Prep Kit (Roche). For whole transcriptome sequencing, RNA samples were depleted of the ribosomal RNA using the Ribo Zero Kit (Illumina) and library preparation was performed using the TruSeq Stranded Total RNA Kit (Qiagen). For small RNA sequencing the QIAseq miRNA Kit (Qiagen) was used All library preparation kits were used according to manufacturer’s instructions. Sequencing was performed on a NovaSeq6000 system (Illumina).
For WGS, average coverage for tumor samples was ≥60X and ≥30X for normal samples with a total genomic coverage of ≥95%.
Whole transcriptome sequencing datasets have ≥100 million total reads with <20% of ribosomal origin and ≥20 million reads mapping to mRNAs according to Ensembl reference. Ribosomal depletion was performed to remove nuclear rRNA and mt-rRNA.

Total RNA was extracted in triplicate using Norgen Total RNA Kit (Norgen, Biotek Corporation, Thorold, ON, Canada) from a pool of mixed cells obtained from the twelve cultures of M-MSCs or of L-MSCs and from the nine cultures of AF-MSCs in triplicate. RNA purity and amounts were measured using a NanoDrop Spectrophotometer (NanoDrop Technologies, INK, Wilmington, DE, USA), whereas RNA integrity (RINA ≥ 8.0) was assessed using an RNA 6000 Nano Kit (Agilent Technologies, CA, USA). miRNA-sequencing libraries were generated with the QiAseq miRNA kit (QIAGEN, Hilden, Germania), assessed by capillary electrophoretic analysis with the Agilent 4200 Tape station and sequenced using 1 × 75 bp-reads on an Illumina NextSeq500 generating about 8 million fragments per sample.