GSM6703283: 3D-Plasma-V1-262; Homo sapiens; miRNA-Seq
Total RNA was extracted using the mirVana PARIS kit and protocol (Thermo Fisher Scientific, catalogue # AM1556), in a randomized order, from either 500μl of plasma kept at −80 °C until extraction. Seventy-five μl of nuclease-free water was used to eluate total RNA which was then precipitated following the method described by Burgos et al. (PMID: 23525801). Briefly, 35 μl of cold (4°C) 7 M ammonium acetate solution (ThermoFisher Scientific, catalogue # 02002268) were added to 70μl of total RNA. This was followed by the addition of 420μl of chilled (−20°C) absolute ethanol (Commercial Alcohols, ON, Canada; catalogue # P006EAAN) and overnight precipitation at −20°C. The mix was then centrifuged at 16 000g at 4°C for 30 min. RNA pellets were then washed twice with 200 μl of cold 80% ethanol followed by centrifugation at 16 000g at 4°C for 5min. It was then dried for 30min at room temperature and 5μl of nuclease-free water was added. The total volume was used for library preparation. Samples were randomized before library preparation. An adapted version of the Truseq Small RNA Sample Prep kit (Illumina, BC, Canada; catalogue # RS-200- 0012) protocol from Burgos et al. (PMID: 23525801) was used to manage low concentrated miRNA samples. In this protocol, half of the standard volumes were used for ligation (3′ and 5′ ends) of RNA sample (5μl), reverse transcription, barcoding (index 1-20: one index per sample) and PCR amplification (15 cycles) to preserve the ratio between RNA amount (which is lower in plasma samples) and the reagents. Purification and size-selection (145-160bp) of the libraries were done by migration on a Novex polyacrylamide TBE Gel, 6% (ThermoFisher Scientific, catalogue # EC6265BOX) followed by elution in 300 μl of nuclease-free water and overnight incubation at 500 RPM at room temperature on an incubating microplate shaker (VWR, ON, Canada, catalogue #12620-930). The libraries were then precipitated according to the manufacturer's instructions (including incubation of the precipitation mix at −80°C for 30min) and were resuspended in 25 μl of 10 mM Tris-HCI pH 8.5 buffer. Library sequencing, including quality control and sample pooling, was performed at the McGill University and Génome Québec Innovation Centre (Québec, Canada). Libraries were first quantified using the Kapa Illumina GA with Revised Primers-SYBR Fast Universal kit (Kapa Biosystems) and the average size fragment was determined using a LabChip GX II (PerkinElmer) instrument. The libraries were normalized and pooled before being denatured in 0.05N NaOH and neutralized using HT1 buffer. The pool was loaded at 225pM on an Illumina NovaSeq S1 lane using Xp protocol as per the manufacturer's recommendations. The run was performed for 1x100 cycles (single-end mode). A phiX library was used as a control and mixed with libraries at 1% level. Base calling was performed with RTA v3. Program bcl2fastq2 v2.20 was then used to demultiplex samples and generate fastq reads. Ten samples were extracted and sequenced twice to account for batch preparation and to confirm standardization.