Bioanalyzer small rna chip

Total RNA was extracted using TRIzol (Invitrogen), with an enhanced overnight −30°C precipitation using glycogen (Sigma) as co-precipitant. RNA quality was checked via Bioanalyzer RNA 6000 Nano chip, while small RNA composition was determined by Bioanalyzer Small RNA chip (both Agilent).

Total RNA extraction from BM-derived EVs was performed using the RNeasy mini extraction kit (Qiagen, Hilden, Germany), according to the manufacturer’s instructions. The MiRNA concentration was measured on a BioAnalyzer 2100 (Agilent, Santa Clara, CA, USA) according to the manufacturer’s instructions. Briefly, the concentrations of the samples to be measured were standardised according to the total RNA concentration, as measured by NanoDrop (ThermoFisher, Waltham, MA, USA), between 20 ng/µL and 50 ng/µL depending on the available sample amount. These were then loaded onto the BioAnalyzer small RNA chip (Agilent, part number 5067-1548) and read by the BioAnalyzer 2100. The resultant miRNA concentration was calculated by multiplying the measured miRNA concentration by the dilution factor of the sample, yielding the miRNA concentration in the undiluted sample.

The quality and integrity of the 40S and 80S RNA extracted were determined on an Agilent Bioanalyzer with the total RNA Nano 6000 Chip. For size selection, RNA was subjected to electrophoresis in 15% urea-polyacrylamide gels (Invitrogen) and fragments of 20-80 nt (40S) or 25-35 nt (80S) in size were excised, with the Agilent small RNA ladder used as a reference. RNA was extracted from the gel pieces by smashing the gels into small pieces with gel smasher tubes and extracting the RNA in 0.5 ml 10 mM Tris pH 7 at 70°C for 10 minutes. The gel pieces were removed and RNA was precipitated in isopropanol. The RNA was then dephosphorylated by incubation with T4 PNK (NEB, M0201S) for 2 hours at 37°C in PNK buffer without ATP. It was precipitated again and purified in isopropanol. Footprints were then analyzed with an Agilent Bioanalyzer small-RNA chip and the Qubit smRNA kit. We used up to 25 ng of footprint RNA as input for library preparation with the SMARTer smRNA-SeqKit for Illumina from Takara/Clontech Laboratories, according to the manufacturer’s instructions. For RNA-seq libraries, total cell RNA was extracted with TRIzol and libraries were prepared with the Illumina TruSeq stranded library preparation kit. Deep-sequencing libraries were sequenced on the Illumina Next-Seq 550 system.

Total RNA was isolated from exosomes using TRIzol LS, according to the manufacturer's instructions, and small RNA concentration was determined using a small RNA Bioanalyzer Chip (Agilent). Preparation and sequencing of the cDNA libraries was performed with 200‐600 ng of small RNA from the total RNA samples, according to the manufacturer's instructions (Illumina). The cDNA sequence library yield was measured using an Agilent 2100 Bioanalyzer (Agilent Technologies), and the samples were pooled in equimolar concentrations for the sequencing run. For sequencing, paired‐end 100 (PE100) cycles were performed on a HiSeq 2000 (Illumina).

Total RNA was isolated from 13 patients (Table 1) using Trizol LS, according to manufacturer's instruction and small RNA concentration was determined using a small RNA Bioanalyzer Chip (Agilent, Santa Clara, CA, USA). Preparation and sequencing of cDNA libraries was performed using 200–600 ng of small RNA from total RNA samples, according to manufacturer's instruction (Illumina, San Diego, CA, USA) and was performed as described previously [24 (link)]. In brief, 13 unique barcode sequences were applied for simultaneous analysis of multiple samples on one lane. The cDNA sequence library yield were measured on an Agilent 2100 Bioanalyzer (Agilent) and the samples were pooled in equimolar concentrations for the sequencing run. Sequencing was performed paired end 100 (PE100) cycles on a HiSeq 2000 (Illumina).